Compositions and methods for treating conditions related to ephrin signaling with cupredoxins

ABSTRACT

The present invention relates to compositions and methods of use of cupredoxins, and variants, derivatives and structural equivalents of cupredoxins that interfere with the ephrin signaling system in mammalian cells. Specifically, the invention relates to compositions and methods that use cupredoxins, such as azurin, rusticyanin and plastocyanin, and variants, derivatives and structural equivalents thereof, to treat cancer in mammals.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §§ 119 and 120 to U.S.Provisional Patent Application Ser. No. 60/764,749, filed Feb. 3, 2006,U.S. Provisional Patent Application Ser. No. 60/682,812, filed May 20,2005, and U.S. patent application Ser. No. 11/244,105, filed Oct. 6,2005, which claims priority to U.S. Provisional Patent Application Ser.No. 60/616,782, filed Oct. 7, 2004, and U.S. Provisional PatentApplication Ser. No. 60/680,500, filed May 13, 2005, and is acontinuation-in-part of U.S. patent application Ser. No. 10/720,603,filed Nov. 24, 2003, which claims priority to U.S. Provisional PatentApplication Ser. No. 60/414,550, filed Aug. 15, 2003, and which is acontinuation-in-part of U.S. patent application Ser. No. 10/047,710,filed Jan. 15, 2002, issued Jul. 12, 2006 as U.S. Pat. No. 7,084,105,which claims priority to U.S. Provisional Patent Application Ser. No.60/269,133, filed Feb. 15, 2001. The entire content of these priorapplications is fully incorporated herein by reference.

STATEMENT OF GOVERNMENTAL INTEREST

The subject matter of this application has been supported by researchgrants from the National Institutes of Health (NIH), Bethesda, Md.,U.S.A., (Grant Numbers A1 16790-21, ES 04050-16, A1 45541, CA09432 andN01-CM97567). The government may have certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to cupredoxins and their use in modulatingcellular functions involving ephrins and ephrin receptors. The inventionalso relates to methods of treating ephrin-related conditions. Moreparticularly, the invention relates to the use of a substantially purecupredoxin in methods of slowing growth and metastasis of cancer cellsand pathological conditions, and specifically those related toephrin/ephrin receptor signaling, as well as other therapeutic methodsrelated to ephrin/ephrin receptor signaling. The invention also relatesto variants, derivatives and structural equivalents of cupredoxins thatretain the ability to interfere with the ephrin signaling system incells.

BACKGROUND

The ephrin receptors (Eph receptors) are a large family of receptortyrosine kinases that regulate a multitude of processes in developingand adult tissues by binding a family of ligands called ephrins. Ephreceptors are divided into either the A- or B-type with ephrin ligands.There are currently nine known members of the A-type, EphA1-8 andEphA10, and four known members of the B-type, EphB1-4 and EphB6. Ingeneral, the A class receptors preferentially bind A-type ligands, whilethe B class receptors preferentially bind the B-type ligands. The Ephreceptors are like other receptor tyrosine kinases, with a singletransmembrane spanning domain, with a glycosylated extracellular regioncomprised of a ligand-binding domain with immunoglobulin-like motifs, acysteine rich region and two fibronectin type III repeats. (Surawska etal., Cytokine & Growth Factor Reviews 15:419-433 (2004)). The ephrinligands are divided into the A and B class depending on their sequenceconservation. EphrinA ligands are glycosylphosphatidylinisotol anchoredand usually bound by Eph-A type receptors, while ephrinB ligands containa transmembrane domain and a short cytoplasmic region and are usuallybound by EphB-type receptors. Id.

The signaling process begins when Eph receptor dimerizes with an ephrinligand, causing the receptor to become phosphorylated. Aggregates ofephrin-EphReceptor complexes are formed by higher-order clustering.Receptor activation is thought to depend on the degree ofmultimerization, but is not limited to the tetrameric form as receptorphosphorylation is observed in both lower- and higher-order forms.Depending on the state of multimerization, distinct Eph receptorcomplexes can induce biological effects. In addition to the “forward”signaling through the Eph receptor into the receptor-expressing cell,there is also “backwards” signaling through the ephrin into theephrin-expressing cell. For example, the cytoplasmic tail on theB-ephrins can become phosphorylated leading to the recruitment ofsignaling effectors and a signal transduction cascade within theephrin-signaling cell. Id.

Ephrins are now known to have roles in many cell-cell interactions,including axon pathfinding, neuronal cell migration, and interactions invascular endothelial cells and specialized epithelia. (Flanagan &Vanderhaeghen, Annu. Rev. Neurosci. 21:309-345 (1998); Frisen et al.,EMBO J. 18:5159-5165 (1999)). Eph receptors have also been implicated ina variety of pathological processes, including tumor progression,pathological forms of angiogenesis, chronic pain following tissuedamage, inhibition of nerve regeneration after spinal cord injury, andhuman congenital malformations. (Koolpe et al., J Biol. Chem.280:17301-17311 (2005)). Eph receptors are also reported to play a rolein the balance of stem cell self-renewal versus cell-fate determinationand differentiation. Id.

Eph receptor and ephrin over-expression can result in tumorigenesis, andare associated with angiogenesis and metastasis in many types of humancancer, including lung, breast and prostate cancer, as well as melanomaand leukemia. (Surawska et al., Cytokine & Growth Factor Reviews15:419-433 (2004)). Over-expression of the Eph receptor is thought notto affect the proliferation of cells, but changes their invasivebehavior. According to one theory, in malignant cells with high levelsof EphA2, the receptors are mislocalized, not able to bind their ephrinligands, and therefore not phosphorylated, resulting in increasedextracellular matrix adhesions and higher metastatic potential.(Ruoslahti, Adv. Cancer Res. 76:1-20 (1999)). Angiogenesis is theformation of new blood vessels and capillaries from pre-existingvasculature and is an essential process for tumor survival and growth.Evidence exists that implicates Eph receptor/ephrin up-regulation duringblood vessel invasion of tumors. (Surawska et al., (2004).) A-typeephrins in particular are associated with tumor angiogenesis, andEphA2-Fc and EphA3-Fc fusion proteins decreased tumor vascular density,tumor volume and cell proliferation, and also increased apoptosis.(Brantley et al., Oncogene 21:7011-7026 (2002)).

The crystal structure of the EphB2 receptor-ephrinB2 complex indicatesthat the ectodomain of the ephrinB2 folding topology is aneight-stranded barrel that is a variation on the common Greek keyP-barrel fold, and shares considerable homology with the cupredoxinfamily of copper-binding proteins, although ephrinB2 does not bindcopper. The main difference between ephrin and the cupredoxin-foldproteins is the unusual length of the ephrin G-H_(L) and C-D_(L) loopswith are part of the dimerization and tetramerization ligand receptorinterfaces, respectively. Crystallization studies further indicate thatthe G-H_(L) loop is involved in receptor binding. (Himanen et al.,Nature 414:933-938 (2001)). The extracellular domain of mouse ephrinB2also has a topological similarity to plant nodulins and phytocyanins.(Toth et al., Developmental Cell, 1:83-92 (2001)).

Reports on regression of cancer in humans and animals infected withmicrobial pathogens date back more than 100 years, originating with theinitial report by Coley. (Clin. Orthop. Relat. Res. 262:3-12 (1891)).Several subsequent reports have shown that microbial pathogens replicateat tumor sites under hypoxic conditions and also stimulate the host'simmune system during infection, leading to an inhibition of cancerprogression. (Alexandrof et al., Lancet 353:1689-1694 (1999); Paglia &Guzman, Cancer Immunol. Immunother. 46:88-92 (1998); Pawelek et al.,Cancer Res. 57:4537-4544 (1997)). Bacterial pathogens such asPseudomonas aeruginosa and many others produce a range of virulencefactors that allow the bacteria to escape host defense and causedisease. (Tang et al., Infect. Immun. 64:37-43 (1996); Clark and Bavoil,Methods in Enzymology, vol. 235, Bacterial Pathogenesis, Academic Press,Inc. San Diego Calif. (1994); Salyers and Whitt, Bacterial Pathogenesis:A Molecular Approach, ASM Press, Washington D.C. (1994)). Some virulencefactors induce apoptosis in phagocytic cells such as macrophages tosubvert host defense. (Monack et al., Proc. Natl. Acad. Sci. USA94:10385-10390 (1997); Zychlinsky and Sansonetti, J. Clin. Investig.100:493-495 (1997)).

Two redox proteins elaborated by P. aeruginosa, the cupredoxin azurinand cytochrome c₅₅₁ (Cyt c₅₅₁), both enter J774 cells and showsignificant cytotoxic activity towards the human cancer cells ascompared to normal cells. (Zaborina et al., Microbiology 146: 2521-2530(2000)). Azurin can also enter human melanoma UISO-Mel-2 or human breastcancer MCF-7 cells. (Yamada et al., PNAS 99:14098-14103 (2002); Punj etal., Oncogene 23:2367-2378 (2004); Yamada et al., Cell. Biol. 7:14181431(2005)). In addition, azurin from P. aeruginosa preferentially entersJ774 murine reticulum cell sarcoma cells, forms a complex with andstabilizes the tumor suppressor protein p53, enhances the intracellularconcentration of p53, and induces apoptosis. (Yamada et al., Infectionand Immunity, 70:7054-7062 (2002)). Azurin also caused a significantincrease of apoptosis in human osteosarcoma cells as compared tonon-cancerous cells. (Ye et al., Ai Zheng 24:298-304 (2003)).

Cytochrome C₅₅₁ (Cyt C₅₅₁) from P. aeruginosa enhances the level oftumor suppressor protein p16^(Ink4a) and inhibits cell cycle progressionin J774 cells. (Hiraoka et al., PNAS 101:6427-6432 (2004)). However,when colon cancer cells, such as HCT 116 cells, or p53-null lung cancerH1299 cells were grown in presence of wild type azurin or wild typecytochrome c₅₅₁ for 3 days, they inhibited the growth of HCT 116 cellsat a much lower concentration (IC50=17 μg/ml for azurin; 12 μg/ml forCyt C) than H1299 cells (>20 μg/ml). Id.

A cancer is a malignant tumor of potentially unlimited growth. It isprimarily the pathogenic replication (a loss of normal regulatorycontrol) of various types of cells found in the human body. Initialtreatment of the disease is often surgery, radiation treatment or thecombination of these treatments, but locally recurrent and metastaticdisease is frequent. Chemotherapeutic treatments for some cancers areavailable but these seldom induce long term regression. Hence, they areoften not curative. Commonly, tumors and their metastases becomerefractory to chemotherapy, in an event known as the development ofmultidrug resistance. In many cases, tumors are inherently resistant tosome classes of chemotherapeutic agents. In addition, such treatmentsthreaten noncancerous cells, are stressful to the human body, andproduce many side effects. Improved agents are therefore needed toprevent the spread of cancer cells.

SUMMARY OF THE INVENTION

The present invention relates to compositions and methods of use ofcupredoxins, and variants, derivatives and structural equivalents ofcupredoxins that interfere with the ephrin signaling system in mammaliancells. Specifically, the invention relates to compositions and methodsthat use cupredoxins, such as azurin and plastocyanin, and variants,derivatives and structural equivalents thereof, to treat cancer inmammals.

One aspect of the invention relates to an isolated peptide that is avariant, derivative or structural equivalent of a cupredoxin, and thatcan inhibit the growth of cancer in mammalian cells or tissues. Thispeptide may be an azurin, plastocyanin, pseudoazurin, plastocyanin,rusticyanin or auracyanin, and specifically an azurin, plastocyanin andrusticyanin. In some embodiments, the cupredoxin is from Pseudomonasaeruginosa, Thiobacillus ferrooxidans, Phormidium laminosum, Alcaligenesfaecalis, Achromobacter xylosoxidan, Bordetella bronchiseptica,Methylomonas sp., Neisseria meningitidis, Neisseria gonorrhoeae,Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylella fastidiosa,Cucumis sativus, Chloroflexus aurantiacus, Vibrio parahaemolyticus orUlva pertusa, and specifically Pseudomonas aeruginosa, Thiobacillusferrooxidans, Phormidium laminosum or Ulva pertusa. The isolated peptidemay be part SEQ ID NOS: 1-17 and 22-23. Additionally, SEQ ID NOS: 1-17and 22-23 may have at least about 90% amino acid sequence identity tothe peptide.

In some embodiments, the isolated peptide may be a truncation of acupredoxin. In specific embodiments, the peptide is more than about 10residues and not more than about 100 residues. The peptide may compriseor, alternatively, consist of P. aeruginosa azurin residues 96-113, P.aeruginosa azurin residues 88-113, Ulva pertusa plastocyanin residues70-84, Ulva pertusa residues 57-98, or SEQ ID NOS: 22-30. In someembodiments, the isolated peptide comprises equivalent residues of asubject cupredoxin as a region of an object cupredoxin selected from thegroup consisting of P. aeruginosa azurin residues 96-113, P. aeruginosaazurin residues 88-113, Ulva pertusa plastocyanin residues 70-84, Ulvapertusa residues 57-98, or SEQ ID NOS: 22-30.

Another aspect of the invention is a composition comprising at least onecupredoxin, or variant, derivative or structural equivalent of acupredoxin that can inhibit the growth of cancer in mammalian cells ortissues in a pharmaceutical composition. In some embodiments, thepharmaceutical composition is formulated for intravenous administration.The cupredoxin may be from Pseudomonas aeruginosa, Thiobacillusferrooxidans, Phormidium laminosum, Alcaligenes faecalis, Achromobacterxylosoxidan, Bordetella bronchiseptica, Methylomonas sp., Neisseriameningitidis, Neisseria gonorrhoeae, Pseudomonas fluorescens,Pseudomonas chlororaphis, Xylella fastidiosa, Cucumis sativus,Chloroflexus aurantiacus, Vibrio parahaemolyticus or Ulva pertusa, andspecifically Pseudomonas aeruginosa, Thiobacillus ferrooxidans,Phormidium laminosum and Ulva pertusa. In some embodiments, thecupredoxin may be SEQ ID NOS: 1-17 and 22-23.

Another aspect of the invention is a method to treat a mammalian patientsuffering a pathological condition related to the ephrin signalingsystem or suffering from cancer, comprising administering to the patienta therapeutically effective amount of a composition comprising at leastone cupredoxin, or variant, derivative or structural equivalent of acupredoxin in a pharmaceutical composition. In some embodiments, thepatient is suffering from interstitial cystitis (IC), lesions associatedwith inflammatory bowel disease (IBD), HIV infection, cardiovasculardisease, central nervous system disorders, peripheral vascular diseases,viral diseases, degeneration of the central nervous system (ChristopherReeve's disease) or Alzheimer's disease. In other embodiments, thepatient is suffering from a cancer, such as breast cancer, liver cancer,gastrointestinal cancer, neuroblastoma, neural cancer, leukemia,lymphoma, prostrate cancer, pancreatic cancer, lung cancer, melanoma,ovarian cancer, endometrial tumor, choriocarcinoma, teratocarcinoma,thyroid cancer, all sarcomas including those arising from soft tissuesand bone, renal carcinomas, epidermoid cancer or non-small cell lungcancer. In some embodiments, the patient is a human.

Another aspect of the invention is a composition comprising at least twoisolated polypeptides that are a cupredoxin, or a variant, derivative orstructural equivalent of a cupredoxin can inhibit the growth of cancerin mammalian cells or tissues. In some embodiments, the composition isin a pharmaceutical composition.

Another aspect of the invention is a kit comprising a composition withat least one cupredoxin, or variant, derivative or structural equivalentof a cupredoxin in a pharmaceutical composition in a vial. The kit maybe designed for intravenous administration.

Another aspect of the invention is a method, comprising contacting themammalian cancer cells with a cupredoxin, or variant, derivative orstructural equivalent thereof; and measuring the growth of the cancercells. The cancer cells may be breast cancer, liver cancer,gastrointestinal cancer, neuroblastoma, neural cancer, leukemia,lymphoma, prostrate cancer, pancreatic cancer, lung cancer, melanoma,ovarian cancer, endometrial tumor, choriocarcinoma, teratocarcinoma,thyroid cancer, all sarcomas including those arising from soft tissuesand bone, renal carcinomas, epidermoid cancer or non-small cell lungcancer. In some embodiments, the cancer cells are in vivo.

Another aspect of the invention is an expression vector which encodes avariant, derivative or structural equivalent of a cupredoxin which caninhibit the growth of cancer in mammalian cells or tissues.

Another aspect of the invention is an isolated peptide that is avariant, derivative or structural equivalent of a cupredoxin; and thatcan bind an ephrin receptor, such as EphA1, EphA2, EphA3, EphA4, EphA5,EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4 and EphB6. Insome embodiments, the peptide also binds an ephrin, such as ephrinA1,ephrinA2, ephrinA3, ephrinA4, ephrinA5, ephrinB1, ephrinB2, ephrinB3 andephrinB4. In other embodiments, the isolated peptide binds an ephrin andits receptor, and specifically Ephrin2B and Eph2B. In a related aspect,an isolated peptide is a variant, derivative or structural equivalent ofa cupredoxin; and that can bind an ephrin, such as ephrinA1, ephrinA2,ephrinA3, ephrinA4, ephrinA5, ephrinB1, ephrinB2, ephrinB3 and ephrinB4.

Another aspect of the invention is a method comprising administering apharmaceutical composition containing at least one cupredoxin, orpeptide of the invention to a mammalian patient to guide the growth ofblood vessels in the patient.

Another aspect of the invention is a method comprising administering apharmaceutical composition containing at least one cupredoxin, orpeptide of the invention to a mammalian patient to decrease the growthof blood vessels in the patient.

Another aspect of the invention is a method comprising administering apharmaceutical composition containing at least one cupredoxin, orpeptide of the invention to a mammalian patient to guide to growth ofneurons in the patient.

Another aspect of the invention is a method comprising administering apharmaceutical composition containing at least one cupredoxin, orpeptide of the invention to a mammalian patient to promote osteogenesisin the patient.

Another aspect of the invention is a method comprising substituting aneffective amount of a pharmaceutical composition containing at least onecupredoxin, or peptide of the invention to a mammalian patient for anephrin in a therapeutic method requiring the use of the ephrin.

Another aspect of the invention is a method comprising administering apharmaceutical composition containing at least one cupredoxin, orpeptide of the invention to a mammalian cell to inhibit the activity ofan ephrin receptor associated with the cell.

Another aspect of the invention is a method comprising administering apharmaceutical composition containing at least one cupredoxin, orpeptide of the invention to increase the activity of an ephrin receptorassociated with the cell.

Another aspect of the invention is a method comprising administering toa human patient having a tissue expressing an ephrin receptor, apharmaceutical composition containing at least one cupredoxin, orpeptide of the invention which comprises a derivative of cupredoxin, ora variant, derivative or structural equivalent fused to a pharmaceuticalagent. In some embodiments, the tissues expressing an ephrin receptor iscancer.

Another aspect of the invention is a method to detect tissues withephrin receptors which has the steps of administering to a human patienta pharmaceutical composition containing at least one cupredoxin, orpeptide of the invention fused to a detectable probe and detecting thedistribution of the probe within the patient.

These and other aspects, advantages, and features of the invention willbecome apparent from the following figures and detailed description ofthe specific embodiments.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1. Amino acid sequence of azurin from Pseudomonas aeruginosa.

SEQ ID NO: 2. Amino acid sequence of plastocyanin from Phormidiumlaminosum.

SEQ ID NO: 3. Amino acid sequence of rusticyanin from Thiobacillusferrooxidans.

SEQ ID NO: 4. Amino acid sequence of pseudoazurin from Achromobactercycloclastes.

SEQ ID NO: 5. Amino acid sequence of azurin from Alcaligenes faecalis.

SEQ ID NO: 6. Amino acid sequence of azurin from Achromobacterxylosoxidans ssp. denitrificans I.

SEQ ID NO: 7. Amino acid sequence of azurin from Bordetellabronchiseptica.

SEQ ID NO: 8. Amino acid sequence of azurin from Methylomonas sp. J.

SEQ ID NO: 9. Amino acid sequence of azurin from Neisseria meningitidisZ2491.

SEQ ID NO: 10. Amino acid sequence of azurin from Neisseria gonorrhoeae.

SEQ ID NO: 11. Amino acid sequence of azurin from Pseudomonasfluorescens.

SEQ ID NO: 12. Amino acid sequence of azurin from Pseudomonaschlororaphis.

SEQ ID NO: 13. Amino acid sequence of azurin from Xylella fastidiosa9a5c.

SEQ ID NO: 14. Amino acid sequence of stellacyanin from Cucumis sativus.

SEQ ID NO: 15. Amino acid sequence of auracyanin A from Chloroflexusaurantiacus.

SEQ ID NO: 16. Amino acid sequence of auracyanin B from Chloroflexusaurantiacus.

SEQ ID NO: 17. Amino acid sequence of cucumber basic protein fromCucumis sativus.

SEQ ID NO: 18. Amino acid sequence of 18-mer azurin peptide, Pseudomonasaeruginosa azurin from residues 96-113.

SEQ ID NO: 19. Amino acid sequence of Pseudomonas aeruginosa azurin fromresidues 88-113.

SEQ ID NO: 20. Amino acid sequence of Ulva pertusa plastocyanin fromresidues 70-84.

SEQ ID NO: 21. Amino acid sequence of Vibrio parahaemolyticus azurin.

SEQ ID NO: 22. Amino acid sequence of Ulva pertusa plastocyanin.

SEQ ID NO: 23. Amino acid sequence of EphrinB2 ectodomain from human.

SEQ ID NO: 24. Amino acid sequence of G-H loop region of human EphrinB2.

SEQ ID NO: 25. Amino acid sequence of the P. aeruginosa azurin regionstructurally analogous to the EphrinB2 G-H loop region.

SEQ ID NO: 26. Amino acid sequence of the Thiobacillus(Acidithiobacillus) ferrooxidans rusticyanin region structurallyanalogous to the EphrinB2 G-H loop region.

SEQ ID NO: 27. Amino acid sequence of the Chloroflexus aurantiacusauracyanin B region structurally analogous to the EphrinB2 G-H loopregion.

SEQ ID NO: 28. Amino acid sequence of the Ulva pertusa plastocyaninregion structurally analogous to the EphrinB2 G-H loop region.

SEQ ID NO: 29. Amino acid sequence of the Cucumis sativus cucumber basicprotein region structurally analogous to the EphrinB2 G-H loop region.

SEQ ID NO: 30. Amino acid sequence of the Cucumis sativus stellacyaninregion structurally analogous to the EphrinB2 G-H loop region.

SEQ ID NO: 31. Amino acid sequence of human EphrinB2 residues 69-138.

SEQ ID NO: 32. Amino acid sequence of Ulva pertusa plastocyanin residues57-98.

SEQ ID NO: 33. Amino acid sequence of human EphrinB2 residues 68-138.

SEQ ID NO: 34. Amino acid sequence of P. aeruginosa azurin residues76-128.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a structural alignment of azurin with ephrinB2. The G-Hloop of ephrin B-2 (region that mediates high-affinity interaction withthe EphB receptors) is indicated by boxes. 1JZG_A is the amino acidsequence of azurin from Pseudomonas aeruginosa (SEQ ID NO: 1). 1KGY_E isthe amino acid sequence of ephrinB2 ectodomain from human (SEQ ID NO:23).

FIG. 2 depicts a structural alignment of cupredoxins with ephrinB2. Thebox indicates the G-H loop of ephrinB2 (15 aa), involved in Eph receptorbinding. Conserved residues are indicated in bold and underlined.Capital letters are super-positions. Dashes are where there are noalignments. The EphrinB2 G-H loop region is SEQ ID NO: 24. The P.aeruginosa azurin region structurally analogous to the EphrinB2 G-H loopregion is SEQ ID NO: 25. The Thiobacillus (Acidithiobacillus)ferrooxidans rusticyanin region structurally analogous to the EphrinB2G-H loop region is SEQ ID NO: 26. The Chloroflexus aurantiacusauracyanin region structurally analogous to the EphrinB2 G-H loop regionis SEQ ID NO: 27. The Phormidium laminosum plastocyanin regionstructurally analogous to the EphrinB2 G-H loop region is SEQ ID NO: 28.The Cucumis sativus cucumber basic protein region structurally analogousto the EphrinB2 G-H loop region is SEQ ID NO: 29. The Cucumis sativusstellacyanin region structurally analogous to the EphrinB2 G-H loopregion is SEQ ID NO: 30.

FIG. 3 depicts a comparison among the structures of the ephrinB2ectodomain from human (1kgy_E), plastocyanin from Ulva pertusa (1 iuz),azurin from Pseudomonas aeruginosa (1jzg_A) and rusticyanin fromThiobacillus (Acidithiobacillus) ferrooxidans (1rcy). In FIG. 3A, thetopology of each protein is shown using TOPS cartoons. TOPS cartoonsrepresent the structure as a sequence of secondary structure elements(SSEs): β-strands (depicted as triangles) and helices (alpha and 310)(depicted as circles), how they are connected in a sequence from aminoto carboxyl terminus, and their relative spatial positions andorientations. The direction of the elements can be deduced from theconnecting lines. “Up” strands are indicated by upward pointingtriangles and “Down” strands by downward pointing triangles. FIG. 3B,the pictures were drawn using the MolMol program (Koradi et al., J. Mol.Graphics. 14:51-55 (1996)).

FIG. 4 depicts a surface plasmon resonance sensorgrams for theassociation of cupredoxins with bound Eph-Fc. Selective binding ofazurin (FIG. 4A), plastocyanin (FIG. 4B), and rusticyanin (FIG. 4C) withEphA-Fc and EphB-Fc proteins is represented.

FIG. 5 depicts a schematic representation of various truncated azurinconstructs derived from full length azurin (SEQ ID NO: 1). Secondarystructure elements are illustrated as arrows for β-sheets and helices(alpha and 310) as rectangles. Various segments of the gene encoding the128 amino acid azurin were fused at the 3′-end of the gst gene (encodingglutathione S-transferase) in frame, cloned in E. coli, hyperexpressedand the fusion proteins purified as described earlier (Yamada et al.,Cell Micro. 7:1418-1431 (2005)).

FIG. 6 depicts the relative binding affinities of azurin and selectivelyconstructed GST-Azu fusions for EphB2-Fc determined in surface plasmonresonance studies. In FIG. 6A, an initial screening experiment wasperformed to determine relative binding strengths of azurin or GST-Azuwherein the SPR traces were recorded after injection of the cupredoxins(100 nM) onto EphB2-Fc-modified CM5 sensor chips. Notably, azurin,GST-Azu 88-113, and GST-Azu 36-128 bind stronger than the native ligand(ephrinB2-Fc) to EphB2-Fc. In FIG. 6B, binding affinity curves for theinteractions of azurin, GST-Azu 88-113, ephrinB2-Fc, and GST-Azu 36-89to immobilized EphB2-Fc after titrating increasing concentrations(0.05-100 nM) of the cupredoxins. Equilibrium resonance signals (Req)were extrapolated from the individual sensorgrams to construct thecurves. Binding dissociation constants (Kd) were calculated (Table 6)after fitting the data to a Langmuir (1:1) binding model using theequation Req=Rmax/(1+Kd/C) and the curve fits are shown connecting thedata points in the titration curves. The quantitative data sets agreewith those from the initial binding screen.

FIG. 7 depicts the binding interactions of azurin and GST-Azu withephrinB2-Fc and EphB2-Fc determined in binding titrations andcompetition assays. In FIG. 7A, SPR binding curves for the interactionsof azurin and GST-Azu with ephrinB2-Fc for which binding affinities (Kd)were determined as previously described. In FIG. 7B, SPR bindingcompetition studies with EphB2-Fc immobilized on CM5 sensor chips.

FIG. 8 depicts the structural alignment comprising the C-terminals ofplastocyanin from Ulva pertusa (1IUZ) and azurin from P. aeruginosa(1JZG_A), (FIG. 8A and FIG. 8B respectively) with human ephrinB2ectodomain (1KGY_E) as computed by the VAST algorithm. Superimposedsecondary structure elements are denoted by a bold capital letter.Dashes and lower-case lettering are where there are no alignments.Secondary structure elements according to the structures are illustratedas arrows for β-sheets and open rectangles for 310 helices. Identicalamino acids are indicated by an asterisk. Amino acids highlighted indark gray and light gray in the ephrin B2 sequence indicate residuesinvolved in the interaction between ephrinB2 and EphB2 receptor and inthe ligand dimerization respectively (Himanen et al., Nature 414:933-938(2001); Toth et al., Dev. Cell. 1:83-92 (2001)). The plastocyanin andazurin peptides (called Plc 70-84 (SEQ ID NO: 20) and Azu 96-113 (SEQ IDNO: 18), respectively), corresponding to the G-H loop region ofephrinB2, which is the main region mediating high affinity binding ofthe ephrins to the Eph receptors are boxed and represented below eachalignment. The G and H loop regions are marked with thick arrows on topof the amino acid sequences of ephrinB2. 1KGY_E residues 69-138 and68-138 are found in SEQ ID NOS: 31 and 33, respectively. 1IUZ residues57-98 is found in SEQ ID NO: 32. 1JZG_A residues 76-128 is found in SEQID NO: 34.

FIG. 9 depicts the effects of cupredoxin peptides on cancer cellviability. (In FIG. 9A, effect of azurin (Azu 96-113) and plastocyanin(Plc 70-84) synthetic peptides on cell viability of AstrocytomaCCF-STTG1 and Glioblastoma LN-229 cancer cell lines. In FIG. 9B, effectof different concentrations of plastocyanin (Plc 70-84) syntheticpeptide on Melanoma UISO-Mel-2 cell viability. Cell viability wasdetermined by MTT assay as described in Example 10. Cancer cells (2×10⁴cells per well in 96-well plates) were treated with the syntheticpeptides at different concentrations for 24 h at 37° C. Data arepresented as the percentage of cell viability as compared to that ofuntreated control (100% viability) In FIG. 9C, cytotoxic activity of Azu96-113 synthetic peptide towards Glioblastoma LN-229 cells. Cytotoxicityeffects were determined by MTT assay. Cancer (2×10⁴ cells per well in96-well plates) were treated with various concentrations of Azu 96-113(10, 25, 50, 75, 100 μM) for 24 h at 37° C. Percent cytotoxicity isexpressed as percentage of cell death as compared to that of untreatedcontrol (0% cytotoxicity).

FIG. 10. Effect of GST-Azu 36-128 and GST-Azu 88-113 on cell viabilityof MCF-7 cells. GST-Azu peptides were added at increasing concentrations(1.25, 6.25 and 12.5 μM) into 96 well plates containing 8×10³ cancercells per well, incubated at 37° C. for 48 h and subsequently analyzedusing MTT assay. GST and GST-Azu 36-89 at the same concentrations anduntreated cells were run in parallel with GST-Azu 36-128 and GST-Azu88-113 as controls.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “cell” includes both the singular or the pluralof the term, unless specifically described as a “single cell.”

As used herein, the terms “polypeptide,” “peptide,” and “protein” areused interchangeably to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid. The terms also apply to naturally occurring aminoacid polymers. The terms “polypeptide,” “peptide,” and “protein” arealso inclusive of modifications including, but not limited to,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation. It will beappreciated that polypeptides are not always entirely linear. Forinstance, polypeptides may be branched as a result of ubiquitination andthey may be circular (with or without branching), generally as a resultof post-translation events, including natural processing event andevents brought about by human manipulation which do not occur naturally.Circular, branched and branched circular polypeptides may be synthesizedby non-translation natural process and by entirely synthetic methods aswell. Further, this invention contemplates the use of both themethionine-containing and the methionine-less amino terminal variants ofthe protein of the invention.

As used herein, the term “pathological condition” includes anatomic andphysiological deviations from the normal that constitute an impairmentof the normal state of the living animal or one of its parts, thatinterrupts or modifies the performance of the bodily functions, and is aresponse to various factors (as malnutrition, industrial hazards, orclimate), to specific infective agents (as worms, parasitic protozoa,bacteria, or viruses), to inherent defects of the organism (as geneticanomalies), or to combinations of these factors.

As used herein, the term “condition” includes anatomic and physiologicaldeviations from the normal that constitute an impairment of the normalstate of the living animal or one of its parts, that interrupts ormodifies the performance of the bodily functions.

As used herein, the term “inhibit cell growth” means the slowing orceasing of cell division and/or cell expansion. This term also includesthe inhibition of cell development or increases cell death.

As used herein, the term “suffering from” includes presently exhibitingthe symptoms of a pathological condition, having a pathologicalcondition even without observable symptoms, in recovery from apathological condition, and recovered from a pathological condition.

A used herein, the term “treatment” includes preventing, lowering,stopping, or reversing the progression or severity of the condition orsymptoms associated with a condition being treated. As such, the term“treatment” includes medical, therapeutic, and/or prophylacticadministration, as appropriate.

A “therapeutically effective amount” is an amount effective to preventor slow the development of, or to partially or totally alleviate theexisting symptoms in a particular condition for which the subject beingtreated. Determination of a therapeutically effective amount is wellwithin the capability of those skilled in the art.

As used herein, the term “deficient in expression of the p53 tumorsuppressor gene” refers to a cell having a p53 tumor suppressor genethat is inactivated, mutated, lost or under produced. For example, sucha deficiency may occur as a result of genetic aberrations within the p53gene or due to epigenic reasons such as hypermethylation of C residuesin the CG islands upstream of the tumor suppressor genes or interactionwith viral and cellular oncogenes.

The term “substantially pure”, when used to modify the term“cupredoxin”, as used herein, refers to a cupredoxin, for example, acupredoxin isolated from the growth medium or cellular contents, in aform substantially free of, or unadulterated by, other proteins and/oractive inhibitory compounds. The term “substantially pure” refers to afactor in an amount of at least about 75%, by dry weight, of isolatedfraction, or at least “75% substantially pure.” More specifically, theterm “substantially pure” refers to a compound of at least about 85%, bydry weight, active compound, or at least “85% substantially pure.” Mostspecifically, the term “substantially pure” refers to a compound of atleast about 95%, by dry weight, active compound, or at least “95%substantially pure.” The substantially pure cupredoxin can be used incombination with one or more other substantially pure compounds orisolated cupredoxins.

The phrases “isolated,” “purified” or “biologically pure” refer tomaterial which is substantially or essentially free from componentswhich normally accompany the material as it is found in its nativestate. Thus, isolated peptides in accordance with the inventionpreferably do not contain materials normally associated with thepeptides in their in situ environment. An “isolated” region refers to aregion that does not include the whole sequence of the polypeptide fromwhich the region was derived. An “isolated” nucleic acid, protein, orrespective fragment thereof has been substantially removed from its invivo environment so that it may be manipulated by the skilled artisan,such as but not limited to nucleotide sequencing, restriction digestion,site-directed mutagenesis, and subcloning into expression vectors for anucleic acid fragment as well as obtaining the protein or proteinfragment in substantially pure quantities.

The term “variant” as used herein with respect to a peptide, refers toamino acid sequence variants which may have amino acids replaced,deleted, or inserted as compared to the wild-type polypeptide. Variantsmay be truncations of the wild-type peptide. Thus, a variant peptide maybe made by manipulation of genes encoding the polypeptide. A variant maybe made by altering the basic composition or characteristics of thepolypeptide, but not at least some of its fundamental activities. Forexample, a “variant” of azurin can be a mutated azurin that retains itsability to inhibit the growth of mammalian cancer cells. In some cases,a variant peptide is synthesized with non-natural amino acids, such asε-(3,5-dinitrobenzoyl)-Lys residues. (Ghadiri & Femholz, J. Am. Chem.Soc., 112:9633-9635 (1990)). In some embodiments, the variant has notmore than 20, 19, 18, 17 or 16 amino acids replaced, deleted or insertedcompared to wild-type peptide. In some embodiments, the variant has notmore than 15, 14, 13, 12 or 11 amino acids replaced, deleted or insertedcompared to wild-type peptide. In some embodiments, the variant has notmore than 10, 9, 8 or 7 amino acids replaced, deleted or insertedcompared to wild-type peptide. In some embodiments, the variant has notmore than 6 amino acids replaced, deleted or inserted compared towild-type peptide. In some embodiments, the variant has not more than 5or 4 amino acids replaced, deleted or inserted compared to wild-typepeptide. In some embodiments, the variant has not more than 3, 2 or 1amino acids replaced, deleted or inserted compared to wild-type peptide.

The term “amino acid,” as used herein, means an amino acid moiety thatcomprises any naturally-occurring or non-naturally occurring orsynthetic amino acid residue, i.e., any moiety comprising at least onecarboxyl and at least one amino residue directly linked by one, two,three or more carbon atoms, typically one (α) carbon atom.

The term “derivative” as used herein with respect to a peptide refers toa peptide that is derived from the subject peptide. A derivationincludes chemical modifications of the peptide such that the peptidestill retains some of its fundamental activities. For example, a“derivative” of azurin can be a chemically modified azurin that retainsits ability to inhibit the growth of mammalian cancer cells. Chemicalmodifications of interest include, but are not limited to, amidation,acetylation, sulfation, polyethylene glycol (PEG) modification,phosphorylation or glycosylation of the peptide. In addition, aderivative peptide maybe a fusion of a polypeptide or fragment thereofto a chemical compound, such as but not limited to, another peptide,drug molecule or other therapeutic or pharmaceutical agent or adetectable probe.

The term “percent (%) amino acid sequence identity” is defined as thepercentage of amino acid residues in a polypeptide that are identicalwith amino acid residues in a candidate sequence when the two sequencesare aligned. To determine % amino acid identity, sequences are alignedand if necessary, gaps are introduced to achieve the maximum % sequenceidentity; conservative substitutions are not considered as part of thesequence identity. Amino acid sequence alignment procedures to determinepercent identity are well known to those of skill in the art. Oftenpublicly available computer software such as BLAST, BLAST2, ALIGN2 orMegalign (DNASTAR) software is used to align peptide sequences. In aspecific embodiment, Blastp (available from the National Center forBiotechnology Information, Bethesda Md.) is used using the defaultparameters of long complexity filter, expect 10, word size 3, existence11 and extension 1.

When amino acid sequences are aligned, the % amino acid sequenceidentity of a given amino acid sequence A to, with, or against a givenamino acid sequence B (which can alternatively be phrased as a givenamino acid sequence A that has or comprises a certain % amino acidsequence identity to, with, or against a given amino acid sequence B)can be calculated as:% amino acid sequence identity=X/Y*100

-   -   where    -   X is the number of amino acid residues scored as identical        matches by the sequence alignment program's or algorithm's        alignment of A and B and    -   Y is the total number of amino acid residues in B.

If the length of amino acid sequence A is not equal to the length ofamino acid sequence B, the % amino acid sequence identity of A to B willnot equal the % amino acid sequence identity of B to A. When comparinglonger sequences to shorter sequences, the shorter sequence will be the“B” sequence. For example, when comparing truncated peptides to thecorresponding wild-type polypeptide, the truncated peptide will be the“B” sequence.

General

Some aspects of the present invention provides compositions and methodsthat use cupredoxins that have structural similarity to ephrin tointerfere with the ephrin signaling system in various mammalian cellsand tissues, and also inhibit the growth of mammalian cancer cells.Specifically, the present invention provides for compositions andmethods that use cupredoxins and variants, derivatives and structuralequivalents thereof, to interfere with the ephrin signaling system, andalso inhibit the growth of mammalian cancer cells in vitro and in vivo.

The inventors previously discovered that pathogenic microorganismssecrete ATP-independent cytotoxic factors, for example redox proteinssuch as azurin from P. aeruginosa, and that such factors cause J774 celldeath by apoptosis, particularly in cancer cells. It was also known thatazurin has a domain from about amino acid residues 50-77 thatfacilitates the protein to enter preferentially into cancer cells toinduce cytotoxicity.

Surprisingly, the inventors have now discovered that a C-terminal domainis found in azurins and other cupredoxins that shows a structuralsimilarity to the ephrins. See, Examples 2, 5 and 9. It is also nowknown that azurin and plastocyanin, and particular regions of thesepeptides that are structurally homologous to the ephrin G-H loop, bindcompetitively to ephrin receptors in a 1:1 ratio. See, Examples 6-8. P.aeruginosa azurin, in particular, binds to ephrin receptors EphB2 andEphA6. See, Example 6. Phormidium laminosum plastocyanin showsspecificity for binding ephrin receptors EphA1, A3 and B2, and to alesser extent ephrin receptors EphA2 and A6. See, Example 6. Finally,rusticyanin shows weak binding to ephrin receptors EphA8 and B1. See,Example 6. It is also now known that azurin region amino acid residues88-113, which contains the structural homology to the ephrin G-H loopregion, binds to ephrin receptor EphB2. See, Example 7. Finally, it isnow known that azurin and the 88-113 region of azurin bind to ephrinB2as well as the ephrin receptor EphB2, and can compete with ephrinB2 forits receptor, EphB2. See, Example 8.

The inventors have also discovered that cupredoxins with regions thathave a structural similarity to the ephrin G-H loop regions inhibit thegrowth of mammalian cancer cells in vitro. In general, Phormidiumlaminosum plastocyanin, Thiobacillus ferrooxidans rusticyanin and P.aeruginosa azurin inhibit the growth in vitro of Mel-2 human melanomacells and MCF-7 human breast cancer cells in a trypan blue assay. See,Example 4. Further, the 88-113 residue region of azurin is now known toinhibit cell growth of MCF-7 breast cancer cells. See, Example 10.Finally, an 18-mer azurin peptide and a 15-mer Ulva pertusa plastocyaninpeptide that correspond to the region of structural similarity to theephrinB2 G-H loop region are now known to inhibit the growth of MCF-7human breast cancer cells, CCF-STTG1 brain tumor astrocytoma cells andLN-229 glioblastoma cells in vitro. See, Examples 3 and 9.

Surprising, cupredoxins with structural similarity to the G-H loop ofephrinB2 also affect ephrin-related development in vivo. It is now knownfrom in vivo studies of ephrin-related development in C. elegans, thatthe cupredoxin rusticyanin interferes with tail muscle formation whilecupredoxin azurin prevents embryonic development, both ephrin-relateddevelopmental processes. See, Example 1.

It is now appreciated that in addition to azurin, the cupredoxinsrusticyanin and plastocyanin also share a structural homology with the Gand H regions of ephrinB2. See, Examples 2 and 5. Further, it is knownthat the azurin and the phytocyanins stellacyanin and cucumber basicprotein also share a significant structural homology with ephrins.Because of the structural conservation in the cupredoxin family ofproteins in general, it is predicted that many other cupredoxins andcupredoxin-like proteins will also display a significant structuralhomology to ephrins. See, Example 2. It is therefore contemplated thatcupredoxin family proteins in general can be used to treat theconditions related to ephrin-signaling, and cancer, using thecompositions and methods of the invention. Specific cupredoxins ofinterest include, but are not limited to, azurin, rusticyanin,plastocyanin, stellacyanin, auracyanin, pseudoazurin and cucumber basicprotein. Exemplary protein sequences are found herein as plastocyanin(SEQ ID NOS: 2 and 22), rusticyanin (SEQ ID NO: 3), pseudoazurin (SEQ IDNO: 4), stellacyanin (SEQ ID NO: 14), auracyanin (SEQ ID NOS: 15 and16), and cucumber basic protein (SEQ ID NO: 17). As used herein, theterm “cupredoxin” refers to any member of the cupredoxin family ofproteins, including cupredoxin-like proteins, such as Laz fromNeisseria.

Azurins are particularly specific, and exemplary protein sequences forthese cupredoxins are found herein, but not limited to, as thoseisolated from Pseudomonas aeruginosa (SEQ ID NO: 1); Alcaligenesfaecalis (SEQ ID NO: 5); Achromobacter xylosoxidans ssp. denitrificans I(SEQ ID NO: 6); Bordetella bronchiseptica (SEQ ID NO: 7); Methylomonassp. J (SEQ ID NO: 8); Neisseria meningitidis (SEQ ID NO: 9); Neisseriagonnorrhoeae (SEQ ID NO: 10), Pseudomonas fluorescens (SEQ ID NO: 11);Pseudomonas chlororaphis (SEQ ID NO: 12); Xylella fastidiosa 9a5c (SEQID NO: 13) and Vibrio parahaemolyticus (SEQ ID NO: 21). In a mostspecific embodiment, the azurin is from Pseudomonas aeruginosa. In otherspecific embodiments, the cupredoxin is plastocyanin (SEQ ID NOS: 2 and22), rusticyanin (SEQ ID NO: 3), pseudoazurin (SEQ ID NO: 4),stellacyanin (SEQ ID NO: 14), auracyanin (SEQ ID NOS: 15 and 16), andcucumber basic protein (SEQ ID NO: 17).

In some embodiments, the cupredoxins have a Greek key beta-barrelstructure. The Greek key beta-barrel structure is a well known proteinfold. See, for example, Zhang & Kim (Proteins 40:409-419 (2000)). TheGreek Key topology was named after a pattern that was common on Greekpottery. It has three up-and-down beta strands connected by hairpins,which are followed by a longer connection to a fourth beta strand, whichlies adjacent to the first beta strand. In a specific embodiment, thecupredoxins and variants, derivatives and structural equivalents ofcupredoxins comprise at least one Greek Key beta-barrel structure. Inanother specific embodiment, the cupredoxins and variants, derivativesand structural equivalents of cupredoxins comprise a Greek Key structureof at least 4 beta strands. In another more specific embodiment, thecupredoxins and variants, derivatives and structural equivalents ofcupredoxins comprise a Greek Key structure of at least eight betastrands. In another specific embodiment, the cupredoxin and variants,derivatives and structural equivalents of cupredoxins comprise more thanone Greek Key beta-barrel structure.

Compositions of the Invention

The invention provides for peptides that are variants, derivatives orstructural equivalents of cupredoxin. In some embodiments, the peptideis substantially pure. In other embodiments, the peptide is in acomposition that comprises, consists of or consists essentially of thepeptide. In other embodiments, the peptide is isolated. In someembodiments, the peptide is less that a full length cupredoxin, andretains some of the functional characteristics of the cupredoxin. Insome embodiments, the peptide retains the ability to interfere withephrin-signaling in mammalian cells and tissues, and/or inhibit thegrowth of mammalian cancer cells. In another specific embodiment, thepeptide does not raise an immune response in a mammal, and morespecifically a human.

The invention also provides compositions comprising at least one, atleast two or at least three cupredoxin(s), or variant(s), derivative(s)or structural equivalent(s) of a cupredoxin. The invention also providescompositions comprising at least one, at least two, or at least threecupredoxin(s) or variant(s), derivative(s) or structural equivalent(s)of cupredoxin(s) in a pharmaceutical composition.

Because of the high structural homology between the cupredoxins, it iscontemplated that other cupredoxins of the family will be able tointerfere with ephrin signaling, and specifically inhibit the growth ofcancer in mammalian cells and tissues. In some embodiments, thecupredoxin is, but is not limited to, azurin, pseudoazurin,plastocyanin, pseudoazurin, rusticyanin or auracyanin. In particularlyspecific embodiments, the azurin is derived from Pseudomonas aeruginosa,Alcaligenes faecalis, Achromobacter xylosoxidans ssp. denitrificans I,Bordetella bronchiseptica, Methylomonas sp., Neisseria meningitidis,Neisseria gonorrhoeae, Pseudomonas fluorescens, Pseudomonaschlororaphis, Xylella fastidiosa 9a5 or Vibrio parahaemolyticus. In avery specific embodiment, the azurin is from Pseudomonas aeruginosa. Inother specific embodiments the cupredoxin is a plastocyanin, and morespecifically a plastocyanin derived from Phormidium laminosum or Ulvapertusa. In other specific embodiments, the cupredoxin in a rusticyanin,and more specifically a rusticyanin derived from Thiobacillusferrooxidans. In other specific embodiments, the cupredoxin comprises anamino acid sequence that is SEQ ID NO: 1-17, 21-22.

The invention provides for amino acid sequence variants of a cupredoxinwhich have amino acids replaced, deleted, or inserted as compared to thewild-type polypeptide. Variants of the invention may be truncations ofthe wild-type polypeptide. As used herein, a “truncation” of apolypeptide is the peptide that results from the removal of at least oneamino acid residue from at least one end of the polypeptide sequence. Insome embodiments, the truncation peptide results from at least theremoval of at least one amino acid reside, at least five amino acidresidues, at least 10 amino acid residues, at least 50 amino acidresidues, or about 100 amino acid residues in total from either or bothends of the polypeptide sequence. In some embodiments, the compositioncomprises a peptide that consists of a region of a cupredoxin that isless that the full length wild-type polypeptide. In some embodiments,the composition comprises a peptide that consists of more than about 10residues, more than about 15 residues or more than about 20 residues ofa truncated cupredoxin. In some embodiments, the composition comprises apeptide that consists of not more than about 100 residues, not more thanabout 50 residues, not more than about 40 residues or not more thanabout 30 residues of a truncated cupredoxin. In some embodiments, thevariant is a peptide to which a cupredoxin, and more specifically to SEQID NOS: 1-17, 21-22 has at least about 90% amino acid sequence identity,at least about 95% amino acid sequence identity or at least about 99%amino acid sequence identity.

In specific embodiments, the variant of cupredoxin comprises P.aeruginosa azurin residues 96-113 (SEQ ID NO: 18), 88-113 (SEQ ID NO:19) or SEQ ID NO: 25. In other embodiments, the variant of cupredoxinconsists of P. aeruginosa azurin residues 96-113 (SEQ ID NO: 18), 88-113(SEQ ID NO: 19) or SEQ ID NO: 25. In other specific embodiments, thevariant of cupredoxin comprises Ulva pertusa residues 70-84 (SEQ ID NO:20), Ulva pertusa residues 57-98 (SEQ ID NO: 32), or Ulva pertusasequence SEQ ID NO: 28. In other specific embodiments, the variant ofcupredoxin consists of Ulva pertusa residues 70-84 (SEQ ID NO: 20), Ulvapertusa residues 57-98 (SEQ ID NO: 32), or Ulva pertusa sequence SEQ IDNO: 28. In other specific embodiments, the variant of cupredoxincomprises Thiobacillus ferrooxidans rusticyanin sequence SEQ ID NO: 26,Chloroflexus aurantiacus auracyanin (SEQ ID NO: 27), Cucumis sativussequences SEQ ID NOS: 29 and 30. In other specific embodiments, thevariant of cupredoxin consists of Thiobacillus ferrooxidans rusticyaninsequence SEQ ID NO: 26, Chloroflexus aurantiacus auracyanin (SEQ ID NO:27), Cucumis sativus sequences SEQ ID NOS: 29 and 30. In other specificembodiments, the variant consists of the equivalent residues to theabove truncated sequences from another cupredoxin. It is alsocontemplated that other cupredoxin variants can be designed that have asimilar activity to any of the aforementioned variants. To do this, thesubject cupredoxin amino acid sequence will be aligned to the objectcupredoxin sequence, such as those that contain the truncated variantsabove, using BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR), the relevantresidues of the truncated variant located on the object cupredoxinsequence, and the equivalent residues found on the subject cupredoxinsequence, and the equivalent truncated variant thus designed.

The variants also include peptides made with synthetic amino acids notnaturally occurring. For example, non-naturally occurring amino acidsmay be integrated into the variant peptide to extend or optimize thehalf-life of the composition in the bloodstream. Such variants include,but are not limited to, D,L-peptides (diastereomer), (Futaki et al., J.Biol. Chem. 276(8):5836-40 (2001); Papo et al., Cancer Res.64(16):5779-86 (2004); Miller et al, Biochem. Pharmacol. 36(1):169-76,(1987)); peptides containing unusual amino acids (Lee et al., J. Pept.Res. 63(2):69-84 (2004)), and olefin-containing non-natural amino acidfollowed by hydrocarbon stapling (Schafineister et al., J. Am. Chem.Soc. 122:5891-5892 (2000); Walenski et al., Science 305:1466-1470(2004)). and peptides comprising ε-(3,5-dinitrobenzoyl)-Lys residues.

In other embodiments, the peptide of the invention is a derivative of acupredoxin. The derivatives of cupredoxin are chemical modifications ofthe peptide such that the peptide still retains some of its fundamentalactivities. For example, a “derivative” of azurin can be a chemicallymodified azurin that retains its ability to interfere withephrin-signaling, and specifically inhibit the growth of cancer inmammalian cells and tissues. Chemical modifications of interest include,but are not limited to, amidation, acetylation, sulfation, polyethyleneglycol (PEG) modification, phosphorylation and glycosylation of thepeptide. In addition, a derivative peptide maybe a fusion of acupredoxin, or variant, derivative or structural equivalent thereof to achemical compound, such as but not limited to, another peptide, drugmolecule or other therapeutic or pharmaceutical agent or a detectableprobe. Derivatives of interest include chemical modifications by whichthe half-life in the bloodstream of the peptides and compositions of theinvention can be extended or optimized, such as by several methods wellknown to those in the art, including but not limited to, circularizedpeptides (Monk et al., BioDrugs 19(4):261-78, (2005); DeFreest et al.,J. Pept. Res. 63(5):409-19 (2004)), N- and C-terminal modifications(Labrie et al., Clin. Invest. Med. 13(5):275-8, (1990)), andolefin-containing non-natural amino acid followed by hydrocarbonstapling (Schafineister et al., J. Am. Chem. Soc. 122:5891-5892 (2000);Walenski et al., Science 305:1466-1470 (2004)).

It is contemplated that the peptides invention may be a variant,derivative and/or structural equivalent of a cupredoxin. For example,the peptides may be a truncation of azurin that has been PEGylated, thusmaking it both a variant and a derivative. In one embodiment, thepeptides of the invention are synthesized with α,α-disubstitutednon-natural amino acids containing olefin-bearing tethers, followed byan all-hydrocarbon “staple” by ruthenium catalyzed olefin metathesis.(Scharmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000); Walenskyet al., Science 305:1466-1470 (2004)). Additionally, peptides that arestructural equivalents of azurin may be fused to other peptides, thusmaking a peptide that is both a structural equivalent and a derivative.These examples are merely to illustrate and not to limit the invention.Variants, derivatives or structural equivalents of cupredoxin may or maynot bind copper.

In another embodiment, the peptide is a structural equivalent of acupredoxin. Examples of studies that determine significant structuralhomology between cupredoxins and other proteins include Toth et al.(Developmental Cell 1:82-92 (2001)). Specifically, significantstructural homology between a cupredoxin and the structural equivalentis determined by using the VAST algorithm. (Gibrat et al., Curr OpinStruct Biol 6:377-385 (1996); Madej et al., Proteins 23:356-3690(1995)). In specific embodiments, the VAST p value from a structuralcomparison of a cupredoxin to the structural equivalent is less thanabout 10⁻³, less than about 10⁻⁵, or less than about 10⁻⁷. In otherembodiments, significant structural homology between a cupredoxin andthe structural equivalent is determined by using the DALI algorithm(Holm & Sander, J. Mol. Biol. 233:123-138 (1993)). In specificembodiments, the DALI Z score for a pairwise structural comparison is atleast about 3.5, at least about 7.0, or at least about 10.0. Examples ofthese determinations are found in Examples 2 and 9.

In some embodiments, the cupredoxin, or variant, derivative orstructural equivalent thereof has some of the functional characteristicsof the P. aeruginosa azurin, Ulva pertusa plastocyanin, Phormidiumlaminosum plastocyanin or Thiobacillis ferrooxidans rusticyanin. In aspecific embodiment, the cupredoxin or variant, derivative or structuralequivalent thereof inhibits interferes with the ephrin-signaling system,and/or inhibits the growth of cancer in mammalian cells and tissues. Theinhibition of growth of mammalian cancer cells or tissues may or may notbe related to any interference with the ephrin-signaling system by thecupredoxin, or variant, derivative or structural equivalent thereof.Methods that determine whether the cupredoxin, or variant, derivative orstructural equivalent thereof interferes with ephrin signaling are wellknown in the art, and include the determining of whether the cupredoxin,or variant, derivative or structural equivalent thereof binds to acomponent of the ephrin signaling pathway, such as, but not limited to,an ephrin and/or an ephrin receptor. The ephrin signaling system at itsbroadest includes the ephrin and associated ephrin receptor, and anymolecules (or “components”) required to transmit the signal bothbackward and forward. In a narrower view, the ephrin signaling systemincludes only the ephrin and related ephrin receptor responsible for thesignaling. The term “interfere” when used in the context of the ephrinsignaling system can result in either an increase or decrease of theassociated ephrin signaling, in either or both of the “forward” and“backward” directions. Methods to measure the interference in the ephrinsignaling system are well known in the art. One method to determineinterference of ephrin signaling is the binding or competition of thepeptide to or with an ephrin or ephrin receptor, or other component ofthe ephrin signaling system, as shown in Examples 6-8. In vivo systemscan be used, such as the C. elegans where it is now known thatcupredoxins can interfere with ephrin signaling to alter tail muscleformation and embryonic development, as described in Example 1. Othermethods include, but are not limited to, the Eph receptorphosphorylation assay in Himanen et al. (Nat. Neurosci. 7:501-509(2004)). and Koolpe et al. (J. Biol. Chem. 280:17301-17311 (2005)).

Because it is now known that cupredoxins and variants of cupredoxinscan, among other things, interfere with ephrin signaling and inhibit thegrowth of cancer in mammalian cells and tissues, it is now possible todesign variants, derivatives and structural equivalents of cupredoxinsthat retain this activity, for example. Such variants, derivatives andstructural equivalents can be made by, for example, creating a “library”of various variants, derivatives and structural equivalents of, and thentesting each for anti-cancer activity, for example, using one of manymethods known in the art, such the exemplary methods in Examples 3, 9and 10. It is contemplated that the resulting variants, derivatives andstructural equivalents of cupredoxins with anti-cancer activity can beused in the methods of the invention, in place of or in addition tocupredoxins.

In other embodiments, the cupredoxin, or variant, derivative orstructural equivalent thereof may have a significant structural homologywith an ephrin. In a specific embodiment, the cupredoxins and variantsand derivative of cupredoxins have a significant structural homologyaround the G-H loop region of ephrin. Examples of studies that determinesignificant structural homology between cupredoxins and ephrins includeToth et al. (Developmental Cell 1:82-92 (2001)). and Example 2 herein.Specifically, significant structural homology between a cupredoxin andan ephrin is determined by using the VAST algorithm. (Gibrat et al.,Curr Opin Struct Biol 6:377-385 (1996); Madej et al., Proteins23:356-3690 (1995)). In specific embodiments, the VAST p value from astructural comparison of a cupredoxin to an ephrin is less than about10⁻³, less than about 10⁻⁵, or less than about 10⁻⁷. In otherembodiments, significant structural homology between a cupredoxin and anephrin is determined by using the DALI algorithm (Holm & Sander, J. Mol.Biol. 233:123-138 (1993)). In specific embodiments, the DALI Z score fora pairwise structural comparison is at least about 3.5, at least about7.0, or at least about 10.0.

In another embodiment, the cupredoxin, or variant, derivative orstructural equivalent thereof binds to either an ephrin and/or an Ephreceptor. It is now known that several cupredoxins have a C-terminalregion that is structurally similar to ephrinB2 ectodomain. See Examples2, 5 and 9. In a specific embodiment, the cupredoxin, or variant,derivative or structural equivalent thereof bind to an ephrin. In aparticularly specific embodiment, the ephrin is, but is not limited to,ephrinA1, ephrinA2, ephrinA3, ephrinA4, ephrinA5, ephrinB1, ephrinB2,ephrinB3 and ephrinB4. In a particularly specific embodiment, the ephrinis, but is not limited to, ephrinB1, ephrinB2, ephrinB3 and ephrinB4. Inanother specific embodiment, the cupredoxin, or variant, derivative orstructural equivalent thereof binds to an Eph receptor. In particularlyspecific embodiments, the Eph receptor is, but is not limited to, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1,EphB2, EphB3, EphB4 and EphB6. In some embodiments, the cupredoxin, orvariant, derivative or structural equivalent thereof binds to both aephrin and an ephrin receptor. In some embodiments, the cupredoxin, orvariant, derivative or structural equivalent thereof binds to a ephrinand its receptor, specifically EphrinB2 and EphB2. Methods fordetermining the binding of proteins to other proteins are well known inthe art. Examples of methods determining binding to Eph receptorsinclude Examples 6-8, as well as Koolpe et al. (J. Biol. Chem.280:17301-17311 (2005)). and Himanen et al. (Nat. Neurosci. 7:501-509(2004)).

In some specific embodiments, the cupredoxin, or variant, derivative orstructural equivalent thereof induces apoptosis in a mammalian cancercell, more specifically a J774 cell. The ability of a cupredoxin orother polypeptide to induce apoptosis may be observed by mitosensorApoAlert confocal microscopy using a MITOSENSOR™ APOLERT™ MitochondrialMembrane Sensor kit (Clontech Laboratories, Inc., Palo Alto, Calif.,U.S.A.), by measuring caspase-8, caspase-9 and caspase-3 activity usingthe method described in Zou et al. (J. Biol. Chem. 274: 11549-11556(1999)), and by detecting apoptosis-induced nuclear DNA fragmentationusing, for example, the APOLERT™ DNA fragmentation kit (ClontechLaboratories, Inc., Palo Alto, Calif., U.S.A.).

In another specific embodiment, the cupredoxin, or variant, derivativeor structural equivalent thereof induces cellular growth arrest in amammalian cancer cell, more specifically a J774 cell. Cellular growtharrest can be determined by measuring the extent of inhibition of cellcycle progression, such as by the method found in Yamada et al. (PNAS101:4770-4775 (2004)). In another specific embodiment, the cupredoxin,or variant, derivative or structural equivalent thereof inhibits cellcycle progression in a mammalian cancer cell, more specifically a J774cell.

Cupredoxins

These small blue copper proteins (cupredoxins) are electron transferproteins (10-20 kDa) that participate in bacterial electron transferchains or are of unknown function. The copper ion is solely bound by theprotein matrix. A special distorted trigonal planar arrangement to twohistidine and one cysteine ligands around the copper gives rise to verypeculiar electronic properties of the metal site and an intense bluecolor. A number of cupredoxins have been crystallographicallycharacterized at medium to high resolution.

The cupredoxins in general have a low sequence homology but highstructural homology. (Gough & Clothia, Structure 12:917-925 (2004); DeRienzo et al., Protein Science 9:1439-1454 (2000).) For example, theamino acid sequence of azurin is 31% identical to that of auracyanin B,16.3% to that of rusticyanin, 20.3% to that of plastocyanin, and 17.3%to that of pseudoazurin. See, Table 1. However, the structuralsimilarity of these proteins is more pronounced. The VAST p value forthe comparison of the structure of azurin to auracyanin B is 10^(−7.4),azurin to rusticyanin is 10⁻⁵, azurin to plastocyanin is 10^(−5.6), andazurin to psuedoazurin is 10^(−4.1).

All of the cupredoxins possess an eight-stranded Greek key beta-barrelor beta-sandwich fold and have a highly conserved site architecture. (DeRienzo et al., Protein Science 9:1439-1454 (2000).) A prominenthydrophobic patch, due to the presence of many long chain aliphaticresidues such as methionines and leucines, is present around the coppersite in azurins, amicyanins, cyanobacterial plastocyanins, cucumberbasic protein and to a lesser extent, pseudoazurin and eukaryoticplastocyanins. Id. Hydrophobic patches are also found to a lesser extentin stellacyanin and rusticyanin copper sites, but have differentfeatures. Id.

TABLE 1 Sequence and structure alignment of azurin (1JZG) from P.aeruginosa to other proteins using VAST algorithm. Alignment % aa PDBlength¹ identity P-value² Score³ RMSD⁴ Description 1AOZ A 2 82 18.310e−7   12.2 1.9 Ascorbate oxidase 1QHQ_A 113 31 10e−7.4 12.1 1.9AuracyaninB 1V54 B 1 79 20.3 10e−6.0 11.2 2.1 Cytocrome c oxidase 1GY2 A92 16.3 10e−5.0 11.1 1.8 Rusticyanin 3MSP A 74 8.1 10e−6.7 10.9 2.5Motile Major Sperm Protein⁵ 1IUZ 74 20.3 10e−5.6 10.3 2.3 Plastocyanin1KGY E 90 5.6 10e−4.6 10.1 3.4 Ephrinb2 1PMY 75 17.3 10e−4.1 9.8 2.3Pseudoazurin ¹Aligned Length: The number of equivalent pairs of C-alphaatoms superimposed between the two structures, i.e. how many residueshave been used to calculate the 3D superposition. ²P-VAL: The VAST pvalue is a measure of the significance of the comparison, expressed as aprobability. For example, if the p value is 0.001, then the odds are1000 to 1 against seeing a match of this quality by pure chance. The pvalue from VAST is adjusted for the effects of multiple comparisonsusing the assumption that there are 500 independent and unrelated typesof domains in the MMDB database. The p value shown thus corresponds tothe p value for the pairwise comparison of each domain pair, divided by500. ³Score: The VAST structure-similarity score. This number is relatedto the number of secondary structure elements superimposed and thequality of that superposition. Higher VAST scores correlate with highersimilarity. ⁴RMSD: The root mean square superposition residual inAngstroms. This number is calculated after optimal superposition of twostructures, as the square root of the mean square distances betweenequivalent C-alpha atoms. Note that the RMSD value scales with theextent of the structural alignments and that this size must be takeninto consideration when using RMSD as a descriptor of overall structuralsimilarity. ⁵ C. elegans major sperm protein proved to be an ephrinantagonist in oocyte maturation (Kuwabara, 2003 “The multifaceted C.elegans major sperm protein: an ephrin signalling antagonist in oocytematuration” Genes and Development, 17:155-161.

Azurin

The azurins are copper-containing proteins of 128 amino acid residueswhich belong to the family of cupredoxins involved in electron transferin certain bacteria. The azurins include those from P. aeruginosa (PA)(SEQ ID NO: 1), A. xylosoxidans, and A. denitrificans. (Murphy et al.,J. Mol. Biol. 315:859-871 (2002)). The amino acid sequence identitybetween the azurins varies between 60-90%, these proteins showed astrong structural homology. All azurins have a characteristic β-sandwichwith Greek key motif and the single copper atom is always placed at thesame region of the protein. In addition, azurins possess an essentiallyneutral hydrophobic patch surrounding the copper site. Id.

Plastocyanins

The plastocyanins are soluble proteins of cyanobacteria, algae andplants that contain one molecule of copper per molecule and are blue intheir oxidized form. They occur in the chloroplast, where they functionas electron carriers. Since the determination of the structure of poplarplastocyanin in 1978, the structure of algal (Scenedesmus, Enteromorpha,Chlamydomonas) and plant (French bean) plastocyanins has been determinedeither by crystallographic or NMR methods, and the poplar structure hasbeen refined to 1.33 Å resolution. SEQ ID NO: 2 shows the amino acidsequence of plastocyanin from Phormidium laminosum, a thermophiliccyanobacterium. SEQ ID NO: 22 shows the amino acid sequence ofplastocyanin from Ulva petrusa.

Despite the sequence divergence among plastocyanins of algae andvascular plants (e.g., 62% sequence identity between the Chlamydomonasand poplar proteins), the three-dimensional structures are conserved(e.g., 0.76 Å rms deviation in the C alpha positions between theChlamydomonas and Poplar proteins). Structural features include adistorted tetrahedral copper binding site at one end of aneight-stranded antiparallel beta-barrel, a pronounced negative patch,and a flat hydrophobic surface. The copper site is optimized for itselectron transfer function, and the negative and hydrophobic patches areproposed to be involved in recognition of physiological reactionpartners. Chemical modification, cross-linking, and site-directedmutagenesis experiments have confirmed the importance of the negativeand hydrophobic patches in binding interactions with cytochrome f, andvalidated the model of two functionally significant electron transferpaths involving plastocyanin. One putative electron transfer path isrelatively short (approximately 4 Å) and involves the solvent-exposedcopper ligand His-87 in the hydrophobic patch, while the other is morelengthy (approximately 12-15 Å) and involves the nearly conservedresidue Tyr-83 in the negative patch. (Redinbo et al., J. Bioenerg.Biomembr. 26:49-66 (1994)).

Rusticyanins

Rusticyanins are blue-copper containing single-chain polypeptidesobtained from a Thiobacillus (now called Acidithiobacillus). The X-raycrystal structure of the oxidized form of the extremely stable andhighly oxidizing cupredoxin rusticyanin from Thiobacillus ferrooxidans(SEQ ID NO: 3) has been determined by multiwavelength anomalousdiffraction and refined to 1.9 Å resolution. The rusticyanins arecomposed of a core beta-sandwich fold composed of a six- and aseven-stranded b-sheet. Like other cupredoxins, the copper ion iscoordinated by a cluster of four conserved residues (His 85, Cys138,His143, Met148) arranged in a distorted tetrahedron. (Walter et al., J.Mol. Biol. 263:730-51 (1996)).

Pseudoazurins

The pseudoazurins are a family of blue-copper containing single-chainpolypeptide. The amino acid sequence of pseudoazurin obtained fromAchromobacter cycloclastes is shown in SEQ ID NO: 4. The X-ray structureanalysis of pseudoazurin shows that it has a similar structure to theazurins although there is low sequence homology between these proteins.Two main differences exist between the overall structure of thepseudoazurins and azurins. There is a carboxy terminus extension in thepseudoazurins, relative to the azurins, consisting of two alpha-helices.In the mid-peptide region azurins contain an extended loop, shortened inthe pseudoazurins, which forms a flap containing a short α-helix. Theonly major differences at the copper atom site are the conformation ofthe MET side-chain and the Met-S copper bond length, which issignificantly shorter in pseudoazurin than in azurin.

Phytocyanins

The proteins identifiable as phytocyanins include, but are not limitedto, cucumber basic protein, stellacyanin, mavicyanin, umecyanin, acucumber peeling cupredoxin, a putative blue copper protein in pea pods,and a blue copper protein from Arabidopsis thaliana. In all exceptcucumber basic protein and the pea-pod protein, the axial methionineligand normally found at blue copper sites is replaced by glutamine.

Auracyanin

Three small blue copper proteins designated auracyanin A, auracyaninB-1, and auracyanin B-2 have been isolated from the thermophilic greengliding photosynthetic bacterium Chloroflexus aurantiacus. The two Bforms are glycoproteins and have almost identical properties to eachother, but are distinct from the A form. The sodium dodecylsulfate-polyacrylamide gel electrophoresis demonstrates apparent monomermolecular masses as 14 (A), 18 (B-2), and 22 (B-1) kDa.

The amino acid sequence of auracyanin A has been determined and showedauracyanin A to be a polypeptide of 139 residues. (Van Dreissche et al;Protein Science 8:947-957 (1999).) His58, Cys123, His128, and Met132 arespaced in a way to be expected if they are the evolutionary conservedmetal ligands as in the known small copper proteins plastocyanin andazurin. Secondary structure prediction also indicates that auracyaninhas a general beta-barrel structure similar to that of azurin fromPseudomonas aeruginosa and plastocyanin from poplar leaves. However,auracyanin appears to have sequence characteristics of both small copperprotein sequence classes. The overall similarity with a consensussequence of azurin is roughly the same as that with a consensus sequenceof plastocyanin, namely 30.5%. The N-terminal sequence region 1-18 ofauracyanin is remarkably rich in glycine and hydroxy amino acids. Id.See exemplary amino acid sequence SEQ ID NO: 15 for chain A ofauracyanin from Chloroflexus aurantiacus (NCBI Protein Data BankAccession No. AAM12874).

The auracyanin B molecule has a standard cupredoxin fold. The crystalstructure of auracyanin B from Chloroflexus aurantiacus has beenstudied. (Bond et al., J. Mol. Biol. 306:47-67 (2001); the contents ofwhich are incorporated for all purposes by reference.) With theexception of an additional N-terminal strand, the molecule is verysimilar to that of the bacterial cupredoxin, azurin. As in othercupredoxins, one of the Cu ligands lies on strand 4 of the polypeptide,and the other three lie along a large loop between strands 7 and 8. TheCu site geometry is discussed with reference to the amino acid spacingbetween the latter three ligands. The crystallographically characterizedCu-binding domain of auracyanin B is probably tethered to theperiplasmic side of the cytoplasmic membrane by an N-terminal tail thatexhibits significant sequence identity with known tethers in severalother membrane-associated electron-transfer proteins. The amino acidsequences of the B forms are presented in McManus et al. (J Biol Chem.267:6531-6540 (1992).). See exemplary amino acid sequence SEQ ID NO: 16for chain B of auracyanin from Chloroflexus aurantiacus (NCBI ProteinData Bank Accession No. 1QHQA).

Stellacyanin

Stellacyanins are a subclass of phytocyanins, a ubiquitous family ofplant cupredoxins. An exemplary sequence of a stellacyanin is includedherein as SEQ ID NO: 14. The crystal structure of umecyanin, astellacyanin from horseradish root (Koch et al., J. Am. Chem. Soc.127:158-166 (2005)). and cucumber stellacyanin (Hart et al., ProteinScience 5:2175-2183 (1996)). is also known. The protein has an overallfold similar to the other phytocyanins. The ephrin B2 protein ectodomaintertiary structure bears a significant similarity to stellacyanin. (Tothet al., Developmental Cell 1:83-92 (2001).) An exemplary amino acidsequence of a stellacyanin is found in the National Center forBiotechnology Information Protein Data Bank as Accession No. 1JER, SEQID NO: 14.

Cucumber Basic Protein

An exemplary amino acid sequence from a cucumber basic protein isincluded herein as SEQ ID NO: 17. The crystal structure of the cucumberbasic protein (CBP), a type I blue copper protein, has been refined at1.8 Å resolution. The molecule resembles other blue copper proteins inhaving a Greek key beta-barrel structure, except that the barrel is openon one side and is better described as a “beta-sandwich” or “beta-taco”.(Guss et al., J. Mol. Biol. 262:686-705 (1996).) The ephrinB2 proteinectodomain tertiary structure bears a high similarity (rms deviation 1.5Å for the 50 α carbons) to the cucumber basic protein. (Toth et al.,Developmental Cell 1:83-92 (2001).)

The Cu atom has the normal blue copper NNSS′ co-ordination with bondlengths Cu—N(His39)=1.93 Å, Cu—S(Cys79)=2.16 Å, Cu—N(His84)=1.95 Å,Cu—S(Met89)=2.61 Å. A disulphide link, (Cys52)-S—S-(Cys85), appears toplay an important role in stabilizing the molecular structure. Thepolypeptide fold is typical of a sub-family of blue copper proteins(phytocyanins) as well as a non-metalloprotein, ragweed allergen Ra3,with which CBP has a high degree of sequence identity. The proteinscurrently identifiable as phytocyanins are CBP, stellacyanin,mavicyanin, umecyanin, a cucumber peeling cupredoxin, a putative bluecopper protein in pea pods, and a blue copper protein from Arabidopsisthaliana. In all except CBP and the pea-pod protein, the axialmethionine ligand normally found at blue copper sites is replaced byglutamine. An exemplary sequence for cucumber basic protein is found inNCBI Protein Data Bank Accession No. 2CBP, SEQ ID NO: 17.

Methods of the Invention

Another aspect of the present invention relates to the use of one ormore cupredoxins and variants, derivatives and structural equivalentsthereof in a method to treat a mammalian patient suffering from apathological condition. More specifically, the condition is related tothe ephrin signaling system. Additionally, the mammalian patient may besuffering from cancer.

In general, it is now known that cupredoxins and variants, derivativesand structural equivalents thereof will interfere with the activity ofthe ephrin signaling system in a cell or tissue. In addition, it is nowknown that cupredoxins and variants, derivatives and structuralequivalents thereof will inhibit the growth of cancer in cells andtissues. In specific embodiments, the cell or tissue is mammalian. Inmore specific embodiments, the cell is human. In some embodiments, thecell is not human. In one embodiment, the cupredoxin and variant,derivative or structural equivalent thereof is administered to a cell toinhibit the activity of an ephrin receptor on the surface of the cell.In another embodiment, the cupredoxin and variant, derivative orstructural equivalent thereof is administered to increase the activityof an ephrin receptor on the surface of the cell. In other specificembodiments, the cupredoxin and variants and derivatives of cupredoxininhibit and/or increase the forward and/or backward ephrin signaling.Further, it is possible, for example, for a forward signal to beinhibited while a backwards signal is increased.

The pathological condition suffered by the mammalian patient isspecifically one that is related to the activity of the ephrin signalingsystem. It is now appreciated that cupredoxins comprise a certainstructural homology to ephrins, and can also act like ephrins in vivo inrelation to the ephrin signaling system. It is contemplated thatcupredoxins can be used to treat pathological conditions that arerelated to the ephrin signaling system. In specific embodiments, thepathological condition is accompanied by a higher than normal or lowerthan normal concentration of a component of the ephrin signaling system.In other specific embodiments, the pathological condition results froman over-expression or under-expression of a component of the ephrinsignaling system. In other specific embodiments, the pathologicalcondition results from the excessive turnover or lack of turnover of acomponent of the ephrin signaling system. In other specific embodiments,the pathological condition causes an over-expression or under-expressionof a component of the ephrin signaling system. In another specificembodiment, the pathological condition causes excessive turnover or lackof turnover of a component of the ephrin signaling system. A “component”of the ephrin signaling system includes any molecule that transmits orcauses to be transmitted a signal resulting from ephrin binding to anephrin receptor. In more specific embodiments, the component is anephrin or an ephrin receptor.

Additionally, the pathological condition can be accompanied by anabnormal distribution, either intracellular, intercellular, ortissue-specific, of a component of the ephrin signaling system. In aspecific embodiment, the ephrin receptor is found in a higherconcentration on the surface of a cell or cells. In a more specificembodiment, an ephrin receptor is up-regulated or down-regulated in atissue. In another more specific embodiment, an ephrin is up-regulatedor down-regulated in a tissue.

In another aspect of the invention, the cupredoxin, or variant,derivative or structural equivalent thereof is administered to a patentwith cancer. In some embodiments, the patient is mammalian, andspecifically human. In other embodiments, the patient is not human.While not limiting the mechanism of action of this treatment method,many cancers are associated with increased or decreased concentrationsof components of ephrin signaling system. Many ephrins and Eph receptorshave been shown to be up-regulated or down-regulated in tumors,particularly in the more aggressive stages of tumor progression. Forexample, EphA2 is up-regulated in breast, liver and prostate cancer, andglioblastoma, esophageal squamous cell cancer, ovarian cancer andmelanoma. In a specific embodiment, the cancer is associated withup-regulated EphA2 receptor. However, it is known that in many cancers,different components of the ephrin signaling system are up-regulated ordown-regulated, in particular, the ephrins and Eph receptors. Examplesof the abnormal expression of ephrins and ephrin receptors in varioustumors are well known in the art, and described in Surawska et al.(Cytokine & Growth Factor Reviews 15:419-433 (2004)). In otherembodiments, the cancer is not associated with abnormal amounts ofcomponents or activities of the ephrin signaling system. In specificembodiments, the cancer is, but is not limited to, breast cancer, livercancer, gastrointestinal cancer, neuroblastoma, neural cancer, leukemia,lymphoma, prostrate cancer, pancreatic cancer, lung cancer, melanoma,ovarian cancer, endometrial tumor, choriocarcinoma, teratocarcinoma,thyroid cancer, all sarcomas including those arising from soft tissuesand bone, renal carcinomas, epidermoid cancer and non-small cell lungcancer. In a specific embodiment, the cancer is deficient in theexpression of p53 tumor suppressor gene.

In other embodiments, the cancer has various attributes related to thestage of the tumor growth. An early step in tumor development isvascularization, where arteries are recruited to supply the tumor withblood. While not limiting the mechanism of operation to any one means,it is known that the ephrin signaling system is related to angiogenesis.In one embodiment, the cancer is one with which angiogenesis isassociated. Another stage of tumor development is metastasis, where thetumor forms metastases, which are defined as tumor implantsdiscontinuous with the primary tumor. While not limiting the mechanismof operation to any one means, it is thought that metastasis is alsorelated to the ephrin signaling system. In one embodiment, the cancer ispre-metastatic. In another embodiment, the cancer is metastatic. Methodof measuring an effect of a compound on angiogenesis are well-known inthe art. Specific methods for measuring angiogenesis include, but arenot limited to, the assays found in the following articles, the contentsof which are incorporated for all purposes by reference: Daniel et al.,Kidney Int. Suppl. 57:S73-S81 (1996); Myers et al., J. Cell Biol.148:343-351 (2000); Pandey et al., Science 268:567-569 (1995); Brantleyet al., Oncogene 21:7011-7026 (2002).

There are many pathological conditions in addition to tumors that areassociated with the ephrin signaling pathway. For example, pathologicalforms of angiogenesis (Adams & Klein, Trends Cardiov. Medicine10:183-188 (2000); Brantley-Sieders & Chen, Angiogenesis 7:17-28 (2004);Noren et al., Proc. Natl. Acad. Sci. USA 101:5583-558 (2004).), chronicpain following tissue damage (Battaglia et al., Nat Neurosci. 6:339-340(2003)), inhibition of nerve regeneration after spinal cord injury(Goldscmit et al., J. Neurosci. 6:339-340 (2003)), and human congenitalmalformations (Twigg et al. Proc. Natl. Acad. Sci. USA 101:8652-8657(2004); Wieland et al., Am. J. Hum. Genet. 74:1209-1215 (2004)), andspecific embodiments of the invention use cupredoxin, or variants,derivatives or structural equivalents thereof to treat thesepathological conditions. In specific embodiments, the pathologicalcondition is interstitial cystitis (IC), lesions associated withinflammatory bowel disease (IBD), HIV infection, cardiovascular disease,central nervous system disorders, peripheral vascular diseases, viraldiseases, degeneration of the central nervous system (ChristopherReeve's disease) and Alzheimer's disease. For methodology related to thetreatment of patients with interstitial cystitis and inflammatory boweldisease, see, for example, U.S. Patent Application Publication20050049176 (published Mar. 3, 2005, the contents of which areincorporated for all purposes by this reference.). For methodologyrelated to the inhibition or stimulation of angiogenesis, see, forexample, U.S. Patent Application Publication No. 20040136983 (publishedJul. 15, 2004, the contents of which are incorporated for all purposesby this reference.). For methodology related to therapies forosteogenesis, see, for example, U.S. Patent Application Publication No.20040265808 (published Jul. 15, 2004, the contents of which areincorporated for all purposes by this reference.).

The cupredoxin or variant, derivative or structural equivalent ofcupredoxin can be administered to the patient by many routes and in manyregimens that will be well known to those in the art. In specificembodiments, the cupredoxin or variant or derivative of cupredoxin isadministered intravenously, intramuscularly, subcutaneously or byinjection into a tumor. In a specific embodiment, cupredoxin or variant,derivative or structural equivalent of cupredoxin is administeredintravenously. In particularly specific embodiments, the cupredoxin orvariant or derivative thereof is administered with chemotherapy topatients with cancer or recovering from cancer.

In addition to pathological conditions, the cupredoxin or variant,derivative or structural equivalent of cupredoxin can be used intherapeutic methods related to other conditions suffered by a patient.Such conditions can be the result of accidents which damage the nervousor vascular system, recovery from other therapies, such as surgery, andconditions related to old age, among others. In one embodiment, thepatient requires the growth or regrowth of blood vessels and thecupredoxin or variant, derivative or structural equivalent of cupredoxinis administered to guide the growth of blood vessels. In anotherembodiment, the patient is in need of a decrease in the growth of bloodvessels and the cupredoxin or variant, derivative or structuralequivalent of cupredoxin is administered to inhibit the growth of bloodvessels. In another embodiment, the cupredoxin or variant, derivative orstructural equivalent of cupredoxin is administered to a patient in needof neuron generation or regeneration to guide the growth of neurons. Inanother embodiment the cupredoxin or variant, derivative or structuralequivalent of cupredoxin is administered to a patient to promoteosteogenesis.

Another aspect of the invention is a method to detect cells that displayspecific Ephrin receptors in vivo. It is now known that the cupredoxinscontain a region of high structural homology to ephrin B2 and otherephrins, and that cupredoxins can bind with specificity to ephrinreceptors in vitro. Therefore, cupredoxins will localize to the surfaceof cells expressing ephrin receptors. Accordingly, in some embodiments,cupredoxins or variants derivatives or structural equivalents ofcupredoxins can be used to locate these Eph receptor-expressing cellsand tissues amongst non-Eph receptor expressing cells or tissues. Inaddition, cupredoxins or variants derivatives or structural equivalentsof cupredoxins that display a binding preference of a particular kind ofEph receptor can be used to locate cells or tissues specificallyexpressing that kind of Eph receptor. In one embodiment, the cupredoxinor variants or derivatives thereof are linked to a detectable probe andadministered to a human patient, and the localization of the detectableprobe is measured in the patient to determine the localization of theEph receptor-expressing cells or tissues. In a particularly specificembodiment, the cell is a cancer cell. The detectable probe may be oneof many currently known in the art. Probes of specific interest, includebut are not limited to, fluorescent probes, radioactive probes, andiodine, gadolinium and gold.

In another embodiment of the invention, the cupredoxins or variantsderivatives or structural equivalents of cupredoxins are linked to adrug and are administered to a human patient in order to deliver thedrug to Eph receptor expressing cells or tissues. In another specificembodiment, the tissue is a tumor. In a particularly specificembodiment, the cell is a cancer cell. The drug may be any kind ofchemical compound that has an effect on a cell expressing Eph receptors.The drug may be an organic or inorganic compound, including, but notlimited to, a peptide, DNA molecule, RNA molecule, a pharmaceuticalcomposition and derivatives of any of these. In specific embodiments,the drug is a toxin such as Pseudomonas exotoxin A domain III, or achemical compound such as taxol, or other drug that kills cancer cells.

A cupredoxin or variant, derivative or structural equivalent ofcupredoxin can be administered to a cell or a human patient byadministering a DNA containing a coding region encoding the cupredoxinor variant, derivative or structural equivalent of cupredoxin operablylinked to a promoter region that is expressed in the desired cell ortissue of the human patient. In a specific embodiment, a DNA encoding acupredoxin or variant, derivative or structural equivalent of cupredoxinis administered to a patient in order to treat a condition amenable totreatment with cupredoxin or variant, derivative or structuralequivalent of cupredoxin. Appropriate vectors and methods to administerthem to cells and human patients are well known in the art.Methodologies to express foreign proteins in human subjects are wellknown in the art and can be adapted to express a cupredoxin or variant,derivative or structural equivalent of cupredoxin in patients. Exemplaryprotocols are found in the following U.S. patents, among other sources:U.S. Pat. No. 6,339,068, issued Jan. 15, 2002; U.S. Pat. No. 6,867,000,issued Mar. 15, 2005; U.S. Pat. No. 6,821,957, issued Nov. 23, 2004;U.S. Pat. No. 6,821,955, issued Nov. 23, 2004, U.S. Pat. No. 6,562,376,issued May 13, 2003; the contents of all of which are incorporated forall purposes by this reference. In more specific embodiments, the DNAencoding a cupredoxin or variant, derivative or structural equivalent ofcupredoxin is injected into a tumor of a patient suffering from cancer.In more specific embodiments, the DNA encoding a cupredoxin or variant,derivative or structural equivalent of cupredoxin is injected into atumor of a patient suffering from cancer.

In some embodiments, the pharmaceutical composition is administered tothe patient by intravenous injection, intramuscular injection,subcutaneous injection, inhalation, topical administration, transdermalpatch, suppository, or oral, and specifically intravenous injection. Thepharmaceutical composition may be administered to the patientsimultaneously or within 1 minute to 1 week or more of theadministration of another drug known to treat specific pathologicalconditions related to ephrin signaling or cancer. The pharmaceuticalcomposition may be administered at about the same time as anotheranti-cancer drug.

Pharmaceutical Compositions Comprising Cupredoxins and Variants,Derivatives and Structural Equivalents of Cupredoxins

Pharmaceutical compositions comprising at least one cupredoxin orvariant, derivative or structural equivalent of cupredoxin can bemanufactured in any conventional manner, e.g., by conventional mixing,dissolving, granulating, dragee-making, emulsifying, encapsulating,entrapping, or lyophilizing processes. The substantially purecupredoxins or variants, derivatives or structural equivalents ofcupredoxins can be readily combined with a pharmaceutically acceptablecarrier well-known in the art. Such carriers enable the preparation tobe formulated as a tablet, pill, dragee, capsule, liquid, gel, syrup,slurry, suspension, and the like. Suitable excipients can also include,for example, fillers and cellulose preparations. Other excipients caninclude, for example, flavoring agents, coloring agents, detackifiers,thickeners, and other acceptable additives, adjuvants, or binders.General methodology on pharmaceutical dosage forms is found in Ansel etal., Pharmaceutical Dosage Forms and Drug Delivery Systems (LippencottWilliams & Wilkins, Baltimore Md. (1999)).

The composition comprising a cupredoxin, or variant, derivative orstructural equivalent thereof used in the invention may be administeredin a variety of ways, including by injection (e.g., intradermal,subcutaneous, intramuscular, intraperitoneal and the like), byinhalation, by topical administration, by suppository, by using atransdermal patch or by mouth. General information on drug deliverysystems can be found in Ansel et al., Id. In some embodiments, thecomposition comprising a cupredoxin, or variant, derivative orstructural equivalent thereof can be formulated and used directly asinjectibles, for subcutaneous and intravenous injection, among others.The composition comprising a cupredoxin, or variant, derivative orstructural equivalent thereof can also be taken orally after mixing withprotective agents such as polypropylene glycols or similar coatingagents.

When administration is by injection, the cupredoxin, or variant,derivative or structural equivalent thereof may be formulated in aqueoussolutions, specifically in physiologically compatible buffers such asHanks solution, Ringer's solution, or physiological saline buffer. Thesolution may contain formulatory agents such as suspending, stabilizingand/or dispersing agents. Alternatively, the cupredoxin, or variant,derivative or structural equivalent thereof may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use. In some embodiments, the pharmaceutical composition does notcomprise an adjuvant or any other substance added to enhance the immuneresponse stimulated by the peptide. In some embodiments, thepharmaceutical composition comprises a substance that inhibits an immuneresponse to the peptide.

When administration is by intravenous fluids, the intravenous fluids foruse administering the cupredoxin, or variant, derivative or structuralequivalent thereof may be composed of crystalloids or colloids.Crystalloids as used herein are aqueous solutions of mineral salts orother water-soluble molecules. Colloids as used herein contain largerinsoluble molecules, such as gelatin. Intravenous fluids may be sterile.

Crystalloid fluids that may be used for intravenous administrationinclude but are not limited to, normal saline (a solution of sodiumchloride at 0.9% concentration), Ringer's lactate or Ringer's solution,and a solution of 5% dextrose in water sometimes called D5W, asdescribed in Table 2.

TABLE 2 Composition of Common Crystalloid Solutions Solution Other Name[Na⁺] [Cl⁻] [Glucose] D5W 5% Dextrose 0 0 252 ⅔ & ⅓ 3.3% Dextrose/ 51 51168 0.3% saline Half-normal 0.45% NaCl 77 77 0 saline Normal saline 0.9%NaCl 154 154 0 Ringer's Ringer's 130 109 0 lactate* solution *Ringer'slactate also has 28 mmol/L lactate, 4 mmol/L K⁺ and 3 mmol/L Ca²⁺.

When administration is by inhalation, the cupredoxin, or variant,derivative or structural equivalent thereof may be delivered in the formof an aerosol spray from pressurized packs or a nebulizer with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin, for use in an inhaler or insulator may be formulatedcontaining a powder mix of the proteins and a suitable powder base suchas lactose or starch.

When administration is by topical administration, the cupredoxin, orvariant, derivative or structural equivalent thereof may be formulatedas solutions, gels, ointments, creams, jellies, suspensions, and thelike, as are well known in the art. In some embodiments, administrationis by means of a transdermal patch. When administration is bysuppository (e.g., rectal or vaginal), cupredoxin or variants,derivatives and structural equivalents thereof compositions may also beformulated in compositions containing conventional suppository bases.

When administration is oral, a cupredoxin, or variant, derivative orstructural equivalent thereof can be readily formulated by combining thecupredoxin, or variant, derivative or structural equivalent thereof withpharmaceutically acceptable carriers well known in the art. A solidcarrier, such as mannitol, lactose, magnesium stearate, and the like maybe employed; such carriers enable the cupredoxin and variants,derivatives and structural equivalents thereof to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a subject to be treated.For oral solid formulations such as, for example, powders, capsules andtablets, suitable excipients include fillers such as sugars, cellulosepreparation, granulating agents, and binding agents.

Other convenient carriers, as well-known in the art, also includemultivalent carriers, such as bacterial capsular polysaccharide, adextran or a genetically engineered vector. In addition,sustained-release formulations that include a cupredoxin, or variant,derivative or structural equivalent thereof allow for the release ofcupredoxin, or variant, derivative or structural equivalent thereof overextended periods of time, such that without the sustained releaseformulation, the cupredoxin, or variant, derivative or structuralequivalent thereof would be cleared from a subject's system, and/ordegraded by, for example, proteases and simple hydrolysis beforeeliciting or enhancing a therapeutic effect.

The half-life in the bloodstream of the compositions of the inventioncan be extended or optimized by several methods well known to those inthe art, including but not limited to, circularized peptides (Monk etal., BioDrugs 19(4):261-78, (2005); DeFreest et al., J. Pept. Res.63(5):409-19 (2004)), D,L-peptides (diastereomer), (Futaki et al., J.Biol. Chem. Feb 23;276(8):5836-40 (2001); Papo et al., Cancer Res.64(16):5779-86 (2004); Miller et al., Biochem. Pharmacol. 36(1):169-76,(1987)); peptides containing unusual amino acids (Lee et al., J. Pept.Res. 63(2):69-84 (2004)), N- and C-terminal modifications (Labrie etal., Clin. Invest. Med. 13(5):275-8, (1990)), and hydrocarbon stapling(Schafineister et al., J. Am. Chem. Soc. 122:5891-5892 (2000); Walenskiet al., Science 305:1466-1470 (2004)). Of particular interest ared-isomerization (substitution) and modification of peptide stability viaD-substitution or L-amino acid substitution.

In various embodiments, the pharmaceutical composition includes carriersand excipients (including but not limited to buffers, carbohydrates,mannitol, proteins, polypeptides or amino acids such as glycine,antioxidants, bacteriostats, chelating agents, suspending agents,thickening agents and/or preservatives), water, oils, saline solutions,aqueous dextrose and glycerol solutions, other pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such as buffering agents, tonicity adjusting agents, wettingagents and the like. It will be recognized that, while any suitablecarrier known to those of ordinary skill in the art may be employed toadminister the compositions of this invention, the type of carrier willvary depending on the mode of administration. Compounds may also beencapsulated within liposomes using well-known technology. Biodegradablemicrospheres may also be employed as carriers for the pharmaceuticalcompositions of this invention. Suitable biodegradable microspheres aredisclosed, for example, in U.S. Pat. Nos. 4,897,268; 5,075,109;5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344 and 5,942,252.

The pharmaceutical compositions may be sterilized by conventional,well-known sterilization techniques, or may be sterile filtered. Theresulting aqueous solutions may be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterilesolution prior to administration.

Administration of Cupredoxins

The cupredoxin, or variant, derivative or structural equivalent thereofcan be administered formulated as pharmaceutical compositions andadministered by any suitable route, for example, by oral, buccal,inhalation, sublingual, rectal, vaginal, transurethral, nasal, topical,percutaneous, i.e., transdermal or parenteral (including intravenous,intramuscular, subcutaneous and intracoronary) administration. Thepharmaceutical formulations thereof can be administered in any amounteffective to achieve its intended purpose. More specifically, thecomposition is administered in a therapeutically effective amount. Inspecific embodiments, the therapeutically effective amount is generallyfrom about 0.01-20 mg/day/kg of body weight.

The compounds comprising cupredoxin, or variant, derivative orstructural equivalent thereof are useful for the treatment of acondition related to ephrin-signaling, or cancer in mammalian cells andtissues, alone or in combination with other active agents. Theappropriate dosage will, of course, vary depending upon, for example,the compound of cupredoxin, or variant, derivative or structuralequivalent thereof employed, the host, the mode of administration andthe nature and severity of the conditions being treated. However, ingeneral, satisfactory results in humans are indicated to be obtained atdaily dosages from about 0.01-20 mg/kg of body weight. An indicateddaily dosage in humans is in the range from about 0.7 mg to about 1400mg of a compound of cupredoxin, or variant, derivative or structuralequivalent thereof conveniently administered, for example, in dailydoses, weekly doses, monthly doses, and/or continuous dosing. Dailydoses can be in discrete dosages from 1 to 12 times per day.Alternatively, doses can be administered every other day, every thirdday, every fourth day, every fifth day, every sixth day, every week, andsimilarly in day increments up to 31 days or more. Alternatively, dosingcan be continuous using patches, i.v. administration and the like.

The method of introducing cupredoxin and/or variant, derivative orstructural equivalent thereof to patients is, in some embodiments,co-administration with other drugs known to treat specific pathologicalconditions related to ephrin signaling or cancer, or other conditions ordiseases Such methods are well-known in the art. In a specificembodiment, the cupredoxin and/or variant, derivative or structuralequivalent thereof are part of an cocktail or co-dosing containing orwith other pathological conditions related to ephrin signaling orcancer. Drugs of interest include those used to treat inflammatory boweldisease, HIV infection, viral diseases, cardiovascular disease,peripheral vascular diseases, central nervous system disorders,degeneration of the central nervous system and Alzheimer's disease.

Drugs for treating with inflammatory bowel disease, include, but are notlimited to, aminosalicylates, such as, sulfasalazine (Azulfidine®),olsalazine (Dipentum®), mesalamine (Asacol®, Pentasa®), and balsalazide(Colazal®); corticosteroids, such as, prednisone, Medrol®,methylprednisolone, hydrocortisone, Budesonide (Entocort EC);immunomodulators, such as, azathioprine (Imuran®), 6-mercaptopurine(6-MP, Purinethol®) and cyclosporine A (Sandimmune®, Neoral®);antibiotics, such as, metronidazole (Flagyl®) and ciprofloxacin(Cipro®); biologic therapies, such as, infliximab (Remicade®); andmiscellaneous therapies, such as, tacrolimus (FK506) and mycophenolatemofetil.

Drugs for treating HIV infection include, but are not limited to,reverse transcriptase inhibitors: AZT (zidovudine [Retrovir®]), ddC(zalcitabine [Hivid®], dideoxyinosine), d4T (stavudine [Zerit®]), and3TC (lamivudine [Epivir®]), nonnucleoside reverse transcriptaseinhibitors (NNRTIS): delavirdine (Rescriptor®) and nevirapine(Viramune®), protease inhibitors: ritonavir (Norvir®), a lopinavir andritonavir combination (Kaletra®), saquinavir (Invirase®), indinavirsulphate (Crixivan®), amprenavir (Agenerase®), and nelfinavir(Viracept®).

Drugs for treating viral diseases include, but are not limited to,acyclovir, varicella zoster immune globulin (VZIG®), peginterferon,ribavirin, acyclovir (Zovirax®), valacyclovir (Valtrex®), famciclovir(Famvir®), amantadine, rimantadine, zanamivir, oseltamivir, and alphainterferon.

Drugs for treating cardiovascular disorders include, but are not limitedto, anticoagulants, antiplatelet agents, thrombolytic agents, adrenergicblockers, adrenergic stimulants, alpha/beta adrenergic blockers,angiotensin converting enzyme (ACE) inhibitors, angiotensin convertingenzyme (ACE) inhibitors with calcium channel blockers, angiotensinconverting enzyme (ACE) inhibitors with diuretics, angiotensin IIreceptor antagonists, calcium channel blockers, diuretics (includingcarbonic anhydrase inhibitors, loop diuretics, potassium-sparingdiuretics, thiazides and related diuretics, vasodilators, vasopressors,etc.

Drugs for treating peripheral vascular disease include, but are notlimited to, pentoxifylline (Trental®), an oral methylxanthinederivative, and cilostazol (Pletal®), a phosphodiesterase III inhibitor;antiplatelet/antithrombotic therapy such as aspirin; anticoagulants suchas heparin and Warfarin® (Coumadin®); cholesterol lowering drugs, suchas, niacin, statins, fibrates, Lopid® tablets (gemfibrozil;Parke-Davis); tricor tablets (fenofibrate; Abbott) bile acidsequestrants, Colestid® tablets (micronized colestipol hydrochloride;Pharmacia and Upjohn); Welchol® tablets (colesevelam hydrochloride;Sankyo); calcium channel blockers; vitamins and dietary supplements,such as, folate, B-6, B-12, L-arginine and omega-3 fatty acids; andHMG-COA Reductase inhibitors, such as, Advicor® tablets(Niacin/Lovastatin; Kos); Altocor® extended-release tablets (lovastatin;Andryx labs); Lescol® capsules (fluvastatin sodium; Novartis & Reliant);Lipitor® tablets (atorvastatin; Parke-Davis and Pfizer); Mevacor®tablets (lovastatin; Merck); Pravachol® tablets (Pravastatin sodium;Bristol-Myers Squibb) Pravigard® PAC tablets (Buffered Aspirin andPravastatin Sodium; Bristol-Myers Squibb); Zocor® tablets (Simvastatin;Merck); nicotinic acid agents, such as, Advicor® tablets(Niacin/Lovastatin; Kos (also listed as a HMG-COA Reductase inhibitor));Niaspan® (niacin; Kos); and miscellaneous agents, such as, Zetia®tablets (ezetimibe; Merck/Schering Plough).

Drugs for treating central nervous system disorders include, but are notlimited to, psychotherapeutic agents, such as, various benzodiazepinepreparations and combinations, antianxiety agents, antidepressants(including monoamine oxidase inhibitors (MAOI), selective serotoninreuptake inhibitors (SSRIs), tricyclic antidepressants), antimanicagents, antipanic agents, antipsychotic agents, psychostimulants, andobsessive-compulsive disorder management agents; migraine preparations,such as, beta adrenergic blocking agents, isometheptene and serotoninreceptor agonists, as well as miscellaneous migraine preparationsincluding active ingredients in Depakote® tablets (Divalproex sodium;Abbott) and Excedrin® migraine tablets (acetaminophen; BMS Products);sedatives and hypnotics; anticonvulsants; and Pimozide®. Drugs fortreating Parkinson's disease include, but are not limited to,anticholinergic agents, catechol-o-methyltransferase inhibitors,dopamine agents and monoamine oxidase (MAO) inhibitors.

Drugs for treating CNS degeneration disorders include the following:Drugs for treating Multiple Sclerosis include, but are not limited to,active ingredients in Avonex® (interferon beta-1a; Biogen Neurology);Betaseron® for SC injection (modified form of Interferon beta-1b;Berlex); Copaxone® for injection (Glatiramer Acetate; TevaNeuroscience); Depo-Medrol® injectable suspension (Methylprednisoloneacetate; Pharmacia & Upjohn); Novantrone® for injection concentrate(Mitoxantrone supplied as mitoxantrone hydrochloride; Serono); Orapred®oral solution (prednisolone sodium phosphate oral solution; Ascent); andRebif® injection (interferon beta-1a; Pfizer & Serono). Drugs fortreating Huntington's Disease include, but are not limited to,tranquilizers such as clonazepam (Klonopin®); antipsychotic drugs suchas haloperidol (Haldol®) and clozapine (Clozaril®); fluoxetine (Prozac®,Sarafem®), sertraline (Zoloft®), nortriptyline (Aventyl®, Pamelor®), andlithium (Eskalith®, Lithobid®).

Drugs for treating Alzheimer's disease include, but are not limited to,Aricept® tablets (Donepezil Hydrochloride; Eisai or Pfizer); Exelon®capsules (rivastigmine (as the hydrogen tartrate salt); Novartis);Exelon® oral solution (rivastigmine tartrate; Novartis); Reminyl® oralsolution (galantamine hydrobromide; Janssen) or Reminyl® tablets(galantamine hydrobromide; Janssen).

The method of introducing compounds comprising a cupredoxin, or variant,derivative or structurally equivalent thereof to patients is, in someembodiments, co-administration with other drugs known to treat cancer.Such methods are well-known in the art. In a specific embodiment, thecompounds containing a cupredoxin, or variant, derivative orstructurally equivalent thereof are part of an cocktail or co-dosingcontaining or with other drugs for treating cancer. Such drugs include,for example, those listed herein and specifically 5-fluorouracil;Interferon α; Methotrexate; Tamoxifen; and Vincrinstine. The aboveexamples are provided for illustration only, many other such compoundsare known to those skilled in the art.

Other drugs suitable for treating cancer include, but not limited to,alkylating agents such as nitrogen mustards, alkyl sulfonates,nitrosoureas, ethylenimines, and triazenes; antimetabolites such asfolate antagonists, purine analogues, and pyrimidine analogues;antibiotics such as anthracyclines, bleomycins, mitomycin, dactinomycin,and plicamycin; enzymes such as L-asparaginase; farnesyl-proteintransferase inhibitors; 5.alpha.-reductase inhibitors; inhibitors of17.beta.-hydroxysteroid dehydrogenase type 3; hormonal agents such asglucocorticoids, estrogens/antiestrogens, androgens/antiandrogens,progestins, and luteinizing hormone-releasing hormone antagonists,octreotide acetate; microtubule-disruptor agents, such as ecteinascidinsor their analogs and derivatives; microtubule-stabilizing agents such astaxanes, for example, paclitaxel (Taxol®), docetaxel (Taxotere®), andtheir analogs, and epothilones, such as epothilones A-F and theiranalogs; plant-derived products, such as vinca alkaloids,epipodophyllotoxins, taxanes; and topiosomerase inhibitors;prenyl-protein transferase inhibitors; and miscellaneous agents such ashydroxyurea, procarbazine, mitotane, hexamethylmelamine, platinumcoordination complexes such as cisplatin and carboplatin; and otheragents used as anti-cancer and cytotoxic agents such as biologicalresponse modifiers, growth factors; immune modulators and monoclonalantibodies. The compounds of the invention may also be used inconjunction with radiation therapy and surgery.

Representative examples of these classes of anti-cancer and cytotoxicagents include but are not limited to mechlorethamine hydrochloride,cyclophosphamide, chlorambucil, melphalan, ifosfamide, busulfan,carmustin, lomustine, semustine, streptozocin, thiotepa, dacarbazine,methotrexate, thioguanine, mercaptopurine, fludarabine, pentastatin,cladribin, cytarabine, fluorouracil, doxorubicin hydrochloride,daunorubicin, idarubicin, bleomycin sulfate, mitomycin C, actinomycin D,safracins, saframycins, quinocarcins, discodermolides, vincristine,vinblastine, vinorelbine tartrate, etoposide, etoposide phosphate,teniposide, paclitaxel, tamoxifen, estramustine, estramustine phosphatesodium, flutamide, buserelin, leuprolide, pteridines, diyneses,levamisole, aflacon, interferon, interleukins, aldesleukin, filgrastim,sargramostim, rituximab, BCG, tretinoin, irinotecan hydrochloride,betamethosone, gemcitabine hydrochloride, altretamine, and topoteca andany analogs or derivatives thereof.

Preferred members of these classes include, but are not limited to,paclitaxel, cisplatin, carboplatin, doxorubicin, caminomycin,daunorubicin, aminopterin, methotrexate, methopterin, mitomycin C,ecteinascidin 743, or pofiromycin, 5-fluorouracil, 6-mercaptopurine,gemcitabine, cytosine arabinoside, podophyllotoxin or podophyllotoxinderivatives such as etoposide, etoposide phosphate or teniposide,melphalan, vinblastine, vincristine, leurosidine, vindesine andleurosine.

Examples of anticancer and other cytotoxic agents useful toco-administer with the compositions of the invention include thefollowing: epothilone derivatives as found in German Patent No.4138042.8; WO 97/19086, WO 98/22461, WO 98/25929, WO 98/38192, WO99/01124, WO 99/02224, WO 99/02514, WO 99/03848, WO 99/07692, WO99/27890, WO 99/28324, WO 99/43653, WO 99/54330, WO 99/54318, WO99/54319, WO 99/65913, WO 99/67252, WO 99/67253 and WO 00/00485; cyclindependent kinase inhibitors as found in WO 99/24416 (see also U.S. Pat.No. 6,040,321); and prenyl-protein transferase inhibitors as found in WO97/30992 and WO 98/54966; and agents such as those described genericallyand specifically in U.S. Pat. No. 6,011,029 (the compounds of which U.S.patent can be employed together with any NHR modulators (including, butnot limited to, those of present invention) such as AR modulators, ERmodulators, with LHRH modulators, or with surgical techniques,especially in the treatment of cancer).

The exact formulation, route of administration, and dosage is determinedby the attending physician in view of the patient's condition. Dosageamount and interval can be adjusted individually to provide plasmalevels of the active cupredoxin, or variant, derivative or structuralequivalent thereof which are sufficient to maintain therapeutic effect.Generally, the desired cupredoxin, or variant, derivative or structuralequivalent thereof is administered in an admixture with a pharmaceuticalcarrier selected with regard to the intended route of administration andstandard pharmaceutical practice.

In one aspect, the cupredoxin, or variant, derivative or structuralequivalent thereof is delivered as DNA such that the polypeptide isgenerated in situ. In one embodiment, the DNA is “naked,” as described,for example, in Ulmer et al., (Science 259:1745-1749 (1993)). andreviewed by Cohen (Science 259:1691-1692 (1993)). The uptake of nakedDNA may be increased by coating the DNA onto a carrier, e.g.,biodegradable beads, which are then efficiently transported into thecells. In such methods, the DNA may be present within any of a varietyof delivery systems known to those of ordinary skill in the art,including nucleic acid expression systems, bacterial and viralexpression systems. Techniques for incorporating DNA into suchexpression systems are well known to those of ordinary skill in the art.See, e.g., WO90/11092, WO93/24640, WO 93/17706, and U.S. Pat. No.5,736,524.

Vectors, used to shuttle genetic material from organism to organism, canbe divided into two general classes: cloning vectors are replicatingplasmid or phage with regions that are essential for propagation in anappropriate host cell and into which foreign DNA can be inserted; theforeign DNA is replicated and propagated as if it were a component ofthe vector. An expression vector (such as a plasmid, yeast, or animalvirus genome) is used to introduce foreign genetic material into a hostcell or tissue in order to transcribe and translate the foreign DNA,such as the DNA of a cupredoxin. In expression vectors, the introducedDNA is operably-linked to elements such as promoters that signal to thehost cell to highly transcribe the inserted DNA. Some promoters areexceptionally useful, such as inducible promoters that control genetranscription in response to specific factors. Operably-linking acupredoxin and variants, derivatives or structural equivalents thereofpolynucleotide to an inducible promoter can control the expression ofthe cupredoxin and/or cytochrome c and variants and derivatives thereofin response to specific factors. Examples of classic inducible promotersinclude those that are responsive to α-interferon, heat shock, heavymetal ions, and steroids such as glucocorticoids (Kaufman, MethodsEnzymol. 185:487-511 (1990)) and tetracycline. Other desirable induciblepromoters include those that are not endogenous to the cells in whichthe construct is being introduced, but, are responsive in those cellswhen the induction agent is exogenously supplied. In general, usefulexpression vectors are often plasmids. However, other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses) arecontemplated.

Vector choice is dictated by the organism or cells being used and thedesired fate of the vector. In general, vectors comprise signalsequences, origins of replication, marker genes, polylinker sites,enhancer elements, promoters, and transcription termination sequences.

Kits Comprising Cupredoxin, or Variant, Derivative or StructuralEquivalent Thereof

In one aspect, the invention provides kits containing one or more of thefollowing in a package or container: (1) a biologically activecomposition comprising at least one cupredoxin, or variant, derivativeor structural equivalent thereof; (2) a biologically active compositioncomprising a co-administered drug; (3) a pharmaceutically acceptableexcipient; (4) a vehicle for administration, such as a syringe; (5)instructions for administration. Embodiments in which two or more ofcomponents (1)-(5) are found in the same packaging or container are alsocontemplated. The co-administered drug may be selected from thosepreviously mentioned.

When a kit is supplied, the different components of the composition maybe packaged in separate containers and admixed immediately before use.Such packaging of the components separately may permit long-term storagewithout losing the active components' functions.

The reagents included in the kits can be supplied in containers of anysort such that the life of the different components are preserved andare not adsorbed or altered by the materials of the container. Forexample, sealed glass ampules may contain lyophilized cupredoxin andvariants, derivatives or structural equivalents thereof, or buffers thathave been packaged under a neutral, non-reacting gas, such as nitrogen.Ampules may consist of any suitable material, such as glass, organicpolymers, such as polycarbonate, polystyrene, etc., ceramic, metal orany other material typically employed to hold similar reagents. Otherexamples of suitable containers include simple bottles that may befabricated from similar substances as ampules, and envelopes, that maycomprise foil-lined interiors, such as aluminum or an alloy. Othercontainers include test tubes, vials, flasks, bottles, syringes, or thelike. Containers may have a sterile access port, such as a bottle havinga stopper that can be pierced by a hypodermic injection needle. Othercontainers may have two compartments that are separated by a readilyremovable membrane that upon removal permits the components to be mixed.Removable membranes may be glass, plastic, rubber, etc.

Kits may also be supplied with instructional materials. Instructions maybe printed on paper or other substrate, and/or may be supplied as anelectronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zipdisc, videotape, audiotape, flash memory device etc. Detailedinstructions may not be physically associated with the kit; instead, auser may be directed to an internet web site specified by themanufacturer or distributor of the kit, or supplied as electronic mail.

Modification of Cupredoxin and Variants, Derivatives or StructuralEquivalents Thereof

A cupredoxin, or variant, derivative or structural equivalent thereofmay be chemically modified or genetically altered to produce variantsand derivatives as explained above. Such variants and derivatives may besynthesized by standard techniques.

In addition to naturally-occurring allelic variants of cupredoxin,changes can be introduced by mutation into cupredoxin coding sequencethat incur alterations in the amino acid sequences of the encodedcupredoxin that do not significantly alter the ability of cupredoxin tointerfere with ephrin signaling or inhibit the growth of cancer. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequences of the cupredoxin without altering biologicalactivity, whereas an “essential” amino acid residue is required for suchbiological activity. For example, amino acid residues that are conservedamong the cupredoxins are predicted to be particularly non-amenable toalteration, and thus “essential.”

Amino acids for which conservative substitutions that do not change theactivity of the polypeptide can be made are well known in the art.Useful conservative substitutions are shown in Table 3, “Preferredsubstitutions.” Conservative substitutions whereby an amino acid of oneclass is replaced with another amino acid of the same type fall withinthe scope of the invention so long as the substitution does notmaterially alter the biological activity of the compound.

TABLE 3 Preferred substitutions Preferred Original residue Exemplarysubstitutions substitutions Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln,Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C) Ser SerGln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro, Ala Ala His (H) Asn, Gln,Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu Norleucine Leu (L)Norleucine, Ile, Val, Met, Ala, Ile Phe Lys (K) Arg, Gln, Asn Arg Met(M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro (P) AlaAla Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp,Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe, Ala, Leu Norleucine

Non-conservative substitutions that affect (1) the structure of thepolypeptide backbone, such as a β-sheet or α-helical conformation, (2)the charge, (3) hydrophobicity, or (4) the bulk of the side chain of thetarget site can modify the cytotoxic factor function. Residues aredivided into groups based on common side-chain properties as denoted inTable 4. Non-conservative substitutions entail exchanging a member ofone of these classes for another class. Substitutions may be introducedinto conservative substitution sites or more specifically intonon-conserved sites.

TABLE 4 Amino acid classes Class Amino acids hydrophobic Norleucine,Met, Ala, Val, Leu, Ile neutral hydrophilic Cys, Ser, Thr acidic Asp,Glu basic Asn, Gln, His, Lys, Arg disrupt chain conformation Gly, Proaromatic Trp, Tyr, Phe

The variant polypeptides can be made using methods known in the art suchas oligonucleotide-mediated (site-directed) mutagenesis, alaninescanning, and PCR mutagenesis. Site-directed mutagenesis (Carter,Biochem J. 237:1-7 (1986); Zoller and Smith, Methods Enzymol.154:329-350 (1987)), cassette mutagenesis, restriction selectionmutagenesis (Wells et al., Gene 34:315-323 (1985)) or other knowntechniques can be performed on the cloned DNA to produce the cupredoxinvariant DNA.

Known mutations of cupredoxins can also be used to create variantcupredoxin to be used in the methods of the invention. For example, theC112D and M44KM64E mutants of Pseudomonas aeruginosa azurin are known tohave cytotoxic and growth arresting activity that is different from thenative azurin, and such altered activity can be useful in the treatmentmethods of the present invention. One embodiment of the methods of theinvention utilize cupredoxin and variants and derivatives thereofretaining the ability to interfere with ephrin signaling or inhibit thegrowth of cancer in mammalian cells. In another embodiment, the methodsof the present invention utilize cupredoxin variants such as theM44KM64E mutant, having the ability to cause cellular growth arrest.

A more complete understanding of the present invention can be obtainedby reference to the following specific Examples. The Examples aredescribed solely for purposes of illustration and are not intended tolimit the scope of the invention. Changes in form and substitution ofequivalents are contemplated as circumstances may suggest or renderexpedient. Although specific terms have been employed herein, such termsare intended in a descriptive sense and not for purposes of limitations.Modifications and variations of the invention as hereinbefore set forthcan be made without departing from the spirit and scope thereof, and,therefore, only such limitations should be imposed as are indicated bythe appended claims.

EXAMPLES Example 1 In Vivo Studies of C. elegans Development

Some ephrins are known in Caenorhabditis elegans to be involved inmuscle formation in the tail and embryonic development. Our preliminaryexperiments indicate that rusticyanin interferes with tail muscleformation (causing paralysis of the tail) while azurin prevents babybirth (embryonic development).

In these experiments, the azurin and rusticyanin genes werehyper-expressed in E. coli. A control E. coli without azurin orrusticyanin genes was maintained. When C. elegans worms were fed controlE. coli for up to 3 days, they were healthy and produced babies. When C.elegans were fed azurin-expressing E. coli, they seemed healthy butproduced very few babies. When C. elegans were fedrusticyanin-expressing E. coli, about 30% could only move their heads orupper parts of their bodies but not their tails or the lower part oftheir body, demonstrating paralysis.

Example 2 Azurin Structural Neighbors Analysis

Protein structure neighbors analysis was determined by direct comparisonof 3-dimensional protein structures in the Molecular Modeling DataBasewith the Vector Alignment Search Tool (VAST) algorithm (Gibrat et al.,Curr Opin Struct Biol 6:377-385 (1996); Madej et al., Proteins23:356-3690 (1995)). The Molecular Modeling DataBase containsexperimental data from crystallographic and NMR structure determinationsand is maintained by the National Center for Biotechnology Information(Bethesda, Md., USA). A program that performs the VAST protein structureneighbors analysis is available through the National Center forBiotechnology Information.

Azurin showed a significant (Vast P value: 10 e-4.6; Alignment length:90 aa; % Identity: 6) (FIG. 1), structural superposition with the MMDB(Molecular Modeling Database) entry 1 KGY E (crystal structure of theEphB2-EphrinB2 complex). The superposition alignment of azurin with thereferred crystal structure is related to the chain E (138 aa), a proteindenominated Ephrin (Ephrin-B2). This computational analysis is inaccordance with the structural data published in Himanen et al. (Nature,vol 414: 933-938 (2001)).

Further VAST analysis indicates that the cupredoxins rusticyanin,auracyanin, plastocyanin, cucumber basic protein and stellacyanin alsoshow significant structural homology to ephrinB2 in the G-H loop region.(FIG. 2)

Example 3 Efficacy of the Synthetic Peptides Derived from Azurin andPlastocyanin

The efficacy of the synthetic peptides derived from azurin andplastocyanin that are structurally similar to the human ephrin B-2 G-Hloop has been analyzed. An 18-mer azurin peptide with the followingsequence has been synthesized by standard techniques:

TFDVSKLKEGEQYMFFCT SEQ ID NO: 18

MCF-7 breast cancer cells were incubated in 16-well plates with 5 and 50ug/ml of the 18-mer azurin peptide for 0, 24, 48 and 72 hours, afterwhich the number of MCF-7 cells were counted in a coulter counter. Thepeptide was seen at 50 ug/ml to inhibit MCF-7 cell growth by 50% in 48to 72 hours, as compared to cells without the synthetic peptidetreatment. The extent of cell growth inhibition was about 25% at 5 ug/mlof the 18-mer synthetic peptide as compared to untreated control. Thisexperiment shows that the synthetic peptide designed solely on itsstructural similarity to the B-2 ephrin does in fact inhibit the cancercell progression promoted by the B-2 ephrin.

Example 4 In Vitro Measurement of Effect of Cupredoxins on the Growth ofMel-2 and MCF-7 Cells

The growth of cells treated with cupredoxins was measured using a16-well plate. Mel-2 or MCF-7 cells (5×10⁵ cells per well) were allowedto adhere to multiwell (16-well, in this instance) plates for 24 hours.After adherence, the growth medium was siphoned off. PBS(phosphate-buffered saline) or various cupredoxins/cytochromes atconcentrations of 0.1 to 10 μM in PBS were then added to the wellscontaining fresh growth media and the growth of the cancer cells wasfollowed for 24, 48 and 72 hours. After the incubation period, trypanblue was added to the culture and the number of dead floating cells wascounted. Both live and dead floating cells were counted to determine theIC50 at various cupredoxin doses. The IC50 is the concentration ofprotein that inhibits the cell culture growth by 50%. At 500,000 cellsper well at 24 hours of growth, enough cells were present forreproducible counts. In the cupredoxin-minus control cell cultures, asthe cells grew, they had less space to adhere to the bottom of the well,began to die and became floating cells. In plastocyanin-treated orrusticyanin-treated cell cultures, the cells also overgrew the surfacearea of the well and began to die and float but their numbers were lessthan the control. However, in the azurin-treated cell cultures, both theMel-2 and MCF-7 cell line growth was inhibited leading to very fewfloating cells.

Example 5 Structural Similarity Between EphrinB2 Ectodomain andCupredoxins

Structural similarities between ephrinB2 ectodomain and cupredoxins weredetermined by using VAST and DALI algorithms (Holm and Sander, J. Mol.Biol. 233:123-138 (1993); Gibrat et al., Curr. Opin. Biol. 6″377-385(1996)). respectively available through the U.S. National Institute ofHeath and the European Bioinformatics Institute. Structure-basedpairwise sequence alignments were calculated using the VAST algorithm.Protein structural diagrams are performed in two dimensions using TOPS(Topology Of Protein Structure) cartoons (Torrance et al.,Bioinformatics 21:2537-2538 (2005)). The assessments of the structureswere performed by using the program Mol Mol (Koradi et al., J. Mol.Graphics 14:51-55 (1996)).

Several homologs were found with both programs (data not shown),including a small subset of monomeric cupredoxin proteins, plastocyanin,azurin and rusticyanin. Specifically, the structural comparison betweenephrinB2 ectodomain and these three cupredoxins (which are chosen asrepresentative proteins of the cupredoxin family), provided VAST andDALI alignments with significant and quite similar scores, respectivelyranging from 11.0 to 9.7 (out of a maximum possible 15.7) and 6.7 to6.4. DALI Z scores <2.0 are structurally dissimilar (Table 5). The mostnotable structural conservation exists between the ephrinB2 ectodomainand plastocyanin, superimposed with a root mean square deviation(r.m.s.d.) of 1.8 Å (calculated over 67 structurally equivalent Cαatoms) (Table 5). Contrastingly, azurin, plastocyanin and rusticyaninexhibit weaker primary sequence identity (less than 10%) with theephrinB2 ectodomain (Table 5).

TABLE 5 Structural similarity of the human ephrin B2 ectodomain to threemembers (plastocyanin, azurin and rusticyanin) of the monodomaincupredoxin family. RMSD^(c) Align- VAST DALI Z to ment % PDB Namescore^(a) score^(b) 1KGY_E length Identity 1IUZ Plastocyanin 11.0 6.41.8 67 9.0 1JZG Azurin 10.1 6.7 3.4 90 5.6 1RCY Rusticyanin 9.7 6.1 3.187 8.0 ^(a)VAST score—The VAST structure-similarity score; this numberis related to the number of secondary structure elements superimposedand the quality of that superposition. Higher VAST scores correlate withhigher similarity. ^(b)DALI Z score—The Z scores are calculated usingpairwise comparisions of the ephrin ectodomain structure with otherstructures in the DALI database. The higher the Z score, the less likelyit is that the similarity between the 3D structures is random (pairswith Z < 2.0 are structurally dissimilar). ^(c)RMSD—Root-mean-squaredeviation of backbone residues in angstroms between the aligned parts ofthe pair of structures.

FIG. 3 shows TOPS cartoons (A) and MolMol pictures (B) of the ephrinB2ectodomain and each of the three cupredoxins under study. Thetopological description showed that the proteins adopt a sandwich of twoβ sheets which form the core of the Greek-key fold and show remarkablestructural similarity in the way of the number and orientation of theβ-strands; 1KGY_E (ephrinB2), 1IUZ (plastocyanin) and 1JZG_A (azurin)(FIG. 3). In contrast, the number and arrangement of α-helices are muchless conserved. In JZG A, the helical structure is unique compared withthe other proteins represented herein. As can be expected for proteinswith different sizes, the loops that connect the elements of secondarystructure showed differences in the lengths and their conformations.1RCY (rusticyanin) has the lowest structural homology, with substantialdifferences in the lengths of shared secondary structure elements (SSEs)and the presence of non-shared SSEs

Example 6 Analysis of Eph-Fc Receptor-Cupredoxin Interactions by SurfacePlasmon Resonance

Specific interactions of the Eph receptors with cupredoxins weredetermined by surface plasmon resonance (SPR) analyses. The humanAstrocytoma CCF-STTG1, Glioblastoma LN 229 and MCF-7 (breast cancer)cells were cultured in RPMI medium 1640 containing 2 mM L-glutamine, 10mM Hepes, 10% (vol/vol) heat-inactivated FBS, 100 units/ml penicillin,and 100 μg/ml streptomycin at 37° C. in a humidifier incubator with 5%CO2 as described earlier (Yamada et al., Cell Microbiol. 7:1418-1431(2005); Hiraoka et al., Proc. Natl. Acad. Sci. USA 101: 6427-6432(2004)). The human melanoma cells of UISO-Mel-2 (Yamada et al., Proc.Natl. Acad. Sci. USA 99:14098-14103 (2002)). were cultivated in MEM withHank's medium supplemented with 10% FBS. Escherichia coli JM109 and BL21(DE3) were used as host strains for hyperproduction of azurin,rusticyanin and plastocyanin. Azurin and rusticyanin were purified asdescribed before (Yamada et al., Proc. Natl. Acad. Sci. USA99:14098-14103 (2002); Yamada et al., Cell Cycle 3:1182-1187 (2004)).Construction and purification of GST-azurin fusion derivatives have beenreported earlier (Yamada et al., Cell Microbiol. 7:1418-1431 (2005)).Purification and expression of plastocyanin from Phormidium laminosumwas essentially carried out as described earlier (Schlarb et al., Gene234:275-283 (1999)), except that E. coli strain BL21(DE3)Cd+(RIL) wasused instead of BL21(DE3). The concentration of fully oxidized proteinwas determined spectrophotometrically at 598 nm, using an extinctioncoefficient of 4700 M⁻¹ cm⁻¹.

The Eph ectodomain Fc fusion proteins and ephrins were purchased aslypholized powders from R & D Systems, Minneapolis, Minn. All otherchemicals used for surface plasmon resonance and growth experiments werepurchased from Biacore AB International or Sigma and were of highanalytical grade. Direct protein-protein interactions betweencupredoxins or GST-peptides with Eph-Fc or ephrinB2-Fc were determinedwith a Biacore X biosensor system (Biacore AB) which is based on surfaceplasmon resonance (SPR) technology.

In initial screening experiments immobilization of azurin, rusticyanin,or plastocyanin to a single channel on the CM5 sensor chip was achievedusing the amine coupling procedure. Sequential injections ofN-hydroxysuccinimide/N-(3-dimethylaminopropyl)-N-ethylcarbodiimide(0.05M/0.2M, 35 μL), cupredoxin protein (255 μM, 50 μL), 1M ethanolamine(50 μL, pH 8.8), and 100 mM NaOH (10 μL) covalently linked the proteinsto CM5 sensor chips with increases in resonance signals of 300 RU.Binding experiments were conducted via sequential injections of 100 nMof Eph-Fc proteins in HBS-EP running buffer (0.01 M HEPES, pH 7.4, 0.15M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20) over the sensor surfacesat flow rates of 5 and 30 μL/min for 2.3 min (70 μL injection) withintermediate injections of 100 mM NaOH (10 μL pulse) to regenerate thecupredoxin-CM5 surface. All binding experiments were run against a bareAu CM5 sensor surface (negative channel) to correct for nonspecificbinding.

The binding screens were conducted on cupredoxin modified sensor chipswith sequential injection of 100 nM of Eph-Fc receptors at a flow rateof 30 μL/min over 2.3 min. The curves represent the beginning of theassociation phase for the interactions of Eph-Fc with azurin,plastocyanin, and rusticyanin. Relative binding affinities were taken asa function of the saturating resonances (Req) which varied from 79 RUfor rusticyanin binding to EphB1-Fc or EphA8-Fc to 1248 RU for azurinbinding to EphB2-Fc. The cross-selective binding of cupredoxins tospecific EphA and EphB receptor proteins is notable with the bestinteractions occurring between cupredoxin and EphB.

SPR sensorgrams for binding of immobilized cupredoxins with Eph-Fcindicated selective recognition between these two subsets of proteins(FIGS. 4A-C). In these measurements, Eph-Fc concentrations were keptconstant at 100 nM so that the differences in the degree of associationexpressed in terms of Req reflect the differences in affinities betweenthe Eph-Fc receptor proteins and cupredoxins. Azurin showed the highestaffinity for EphB2-Fc and A6 and also tightly bound A4 and A7 (FIG. 4A).On the other hand, plastocyanin showed selectivity for EphA1-Fc, A3 andB2 and, to a lesser degree, A2 and A6 (FIG. 4B). Lastly, rusticyaninrecognized EphA8-Fc, and B1, but only weakly (FIG. 4C). The associativeinteractions of azurin with EphB2-Fc and EphA6-Fc were the highest withReq values 1248 and 1200 RU respectively.

Example 7 Analysis of Binding Affinities of Azurin and GST-Azu Peptideswith EphB2-Fc-SPR Binding Measurements

Binding affinities of azurin and GST-azu peptides with EphB2-Fc-SPRbinding measurements were performed with azurin or GST-Azu peptidesusing the immobilized EphB2-Fc to evaluate the relative binding affinityof the full length azurin or its various domains for the EphB2-Fcreceptor. Azurin or GST-Azu peptides were injected at increasingconcentrations (0.05-100 nM) to EphB2-Fc or ephrinB2-Fc modified CM5chips with 100 mM NaOH pulses in between injections. The data were fitto a Langmuir (1:1) binding model [Req=Rmax/(1+Kd/C) to extrapolateequilibrium binding constants (Kd).

The experimental strategy for elucidating the structural determinant ofazurin for EphB2-Fc binding is shown in FIG. 5 which depicts the map ofthe primary/secondary structural parameters of azurin and the GST-Azuconstructs as described earlier (Yamada et al., Cell Microbiol.7:1418-1431 (2005)). In particular, the GST-Azu 88-113 is coincidentwith the GH loop region of ephrinB2 (native ligand) and therefore itsbinding efficacy with EphB2-Fc was of particular interest. Initialbinding measurements of azurin and various GST-Azu constructs (all at100 nM) for EphB2-Fc revealed their relative affinity: i.e.,azurin>GST-Azu 88-113>GST-Azu 36-128>ephrinB2-Fc>GST-Azu 36-89>>GST(FIG. 6A). Azurin and GST-tagged azurin showed comparable affinities(data not shown). To quantify the binding affinities, the saturatingresponse values (Req) for azurin or GST-Azu were measured as a functionof their concentrations (0-100 nM) (FIG. 6B). The binding data were fitto a simple Langmuir (1:1) binding equation to determine equilibriumdissociation constants (Kd). Notably, azurin (Kd=6 nM) and GST-Azu88-113 (Kd=12 nM) had 5- and 2.5-fold higher affinities, respectively,for EphB2-Fc than its native ephrinB2-Fc ligand (Kd=30 nM). Also,GST-Azu 36-128 (Table 6) showed slightly higher affinity (Kd=23 nM) thanephrinB2-Fc (Kd=30 nM). In contrast, GST-Azu 36-89 and GST exhibitednegligible binding (Table 6). These data indicate the significance ofthe Azu 88-113 region of azurin for high affinity EphB2-Fc interaction.The Azu 88-113 region consists of the GH loop domain that isstructurally homologous to the GH loop found in ephrinB2.

TABLE 6 Relative binding affinities of azurin and GST-Azu constructswith EphB2-Fc Analyte R_(eq) in RU Molecular Weight K_(d) in nM wtazurin 427 13,929  6 ± 0.75 ephrinB2-Fc 159 130,200 30 ± 5.1 GST 1426,000 >160 GST-Azu 36-128 244 36,076 23 ± 1.5 GST-Azu 36-89 57 31,92461 ± 9.0 GST-Azu 88-113 314 28,943 12 ± 1.5

The relative binding strengths of azurin and GST-Azu constructs withEphB2-Fc were determined in SPR binding titration experiments and theextrapolated datasets are summarized here and are compared to thebinding of native ephrinB2-Fc ligand and GST. The data from an initialscreening experiment were generated upon injection of 100 nM of analyteto the sensor surfaces and the saturating signals (R_(eq)) are listed inresonance units. Saturation in the binding was achieved at eachtitration point in the binding curves depicted in FIG. 6 b and thesevalues plotted against [analyte] were used to calculate equilibriumdissociation constants (K_(d)). K_(d) values ranged from 6 to 61 nM forthe interactions of azurin and GST-Azu while EphB2-Fc with the nativeligand had an intermediate binding affinity within this range.

Example 8 Analysis of Competition Binding Studies for Azurin/GST-Azu andEphrinB2-Fc with EphB2-Fc

To better understand the physiological effects for the high-affinityazurin-EphB2-Fc binding, we performed further binding measurements.Competition binding titrations were conducted similar to the bindingconstant studies except that ephrinB2-Fc (246 nM)+competitor [azurin orGST-Azu (0-1020 nM)] were injected over the EphB2-Fc CM5 sensor surface.

Azurin+ephrinB2-Fc samples were added at different azurin concentrations(0-1020 nM, [ephrinB2-Fc] is 246 nM) to the sensor surface and the datawere plotted as a ratio of resonances, % total response[Req(azurin+ephrinB2-Fc)/(Req/(ephrinB2-Fc)).]. GST-Azu 88-113 andGST-Azu 36-89 were titrated with ephrinB2-Fc to immobilized EphB2-Fc andanalyzed in a similar manner. Competition data suggest 1:1 stoichiometryof binding between azurin and GST-Azu 88-113 with immobilized EphB2-Fc.

We first measured the binding of azurin and GST-Azu constructs to theEphB2-Fc ligand, ephrinB2-Fc. FIG. 7A shows that azurin indeed bindsephrinB2-Fc with high affinity (Kd=8.5+0.8 nM), reflecting thestructural similarities between these two proteins (Table 5). Relativehigh affinity of GST-Azu 88-113 (Kd=39+6.5 nM) for ephrinB2-Fc indicatesthat the region between 88 and 113 is also responsible for binding toephrinB2-Fc. In contrast, GST and GST-Azu 36-89 showed no significantinteractions with ephrinB2-Fc. Taken together, these data indicate thatazurin can bind with high affinity to both EphB2-Fc and ephrinB2-Fc viaits GH loop (88-113) region.

To further test this notion, we performed SPR analysis of binding ofephrinB2-Fc to EphB2-Fc immobilized onto the CM5 sensor chip afterephrinB2-Fc is incubated with varying concentrations of azurin andGST-Azu constructs. FIG. 7B shows the inhibition of ephrinB2-Fc([ephrinB2-Fc]=246 nM) binding to EphB2-Fc by 0-1020 nM of azurin,GST-Azu 88-113, and GST-Azu 36-89, respectively. The inhibition isexpressed in terms of % total response(=[Req(azurin+ephrinB2-Fc)/Req(ephrinB2-Fc alone)]*100). The inhibitionprofiles indicate diminished binding of total protein to the immobilizedEphB2-Fc when ephrinB2-Fc is preincubated with azurin or GST-Azu 88-113,with azurin being a more potent inhibitor than GST-Azu 88-113. Thepreincubation of azurin or GST-Azu 88-113 with ephrinB2-Fc reduced thetotal protein binding to the surface by up to 60%. GST-Azu 36-89, on theother hand, did not affect total protein binding (FIG. 7B), and this isconsistent with its weak binding to either ephrinB2-Fc or EphB2-Fc. Itappears that azurin (or GST-Azu 88-113) forms a stoichiometric complexwith ephrinB2-Fc because maximal inhibition was achieved with 1 to 1ratio of ephrinB2-Fc and azurin (or GST-Azu 88-113). The fact that theinhibition by azurin or GST-Azu 88-113 levels off at 40 to 50% indicatesthat the putative azurin-ephrinB2-Fc complex has some affinity for theEphB2-Fc receptor. Collectively, these binding data indicate that azurinhas high affinity for both ephrinB2-Fc and EphB2-Fc and that it caninterfere with ephrinB2-Fc-EphB2-Fc binding by a dual mechanism ofligand sequestration and receptor occupation.

Example 9 Analysis of Cytotoxic Activity of Azu 96-113 and Plc70-84Synthetic Peptides and GST-Azu Fusion Derivatives Toward Various CancerCell Lines

Upon structure-based sequence alignment of azurin and plastocyanin withhuman ephrinB2 ectodomain, we designed the peptides corresponding to theG-H loop region of ephrinB2 (called Azu 96-113 (SEQ ID NO: 18) and Plc70-84 (SEQ ID NO: 20)), which is the main region mediating high affinitybinding of the ephrins to the Eph receptors (Himanen et al., Nature414:933-938 (2001); Toth et al., Dev. Cell. 1:83-92 (2001)). In FIG. 8,the structural superimposition of the C-terminal segments of ephrinB2ectodomain, azurin and plastocyanin can be seen. Structurally basedsequence alignment of azurin and plastocyanin with human ephrinB2ectodomain was used to design peptides based on the region mediatinghigh affinity binding of the ephrinB2 to the EphB2 receptor (Gstrand-loop-H strand of the ephrinB2-Fc ectodomain) of azurin, namelyAzu 96-113 (96-TFDVSKLKEGEQYMFFCT-113) (SEQ ID NO: 18) and plastocyanin,namely Plc 70-84 (70-VRKLSTPGVYGVYCE-84) (SEQ ID NO: 20) (FIG. 8). Thepeptides were purchased from GenScript Corporation (Piscataway, N.J.) as99% pure. They were purified by reverse phase high-pressure liquidchromatography and their identity verified by mass spectrometry.Peptides were dissolved in phosphate-buffered saline (PBS) (1×) andstored in aliquots at −20° C. until use.

In order to see if such domains of azurin may also play a role asantagonists to Eph signaling in cancer progression, we performedquantitative MTT assays in a number of cancer cell lines normally knownto hyperexpress EphB receptors/ephrin ligands. During a 24 h incubationat 37° C., both azurin and plastocyanin G-H loop peptides at 75 μMshowed induction of cell death in brain tumors astrocytoma CCF-STTG1 andglioblastoma LN-229 (FIG. 9A). The plastocyanin peptide Plc 70-84 showedsomewhat higher cytotoxic activity than the azurin peptide Azu 96-113.To determine if such cytotoxicity is dose dependent and effective forother cancer cell lines, we evaluated the effect of severalconcentrations of these peptides on melanoma (FIG. 9B) or glioblastomacells (FIG. 9C). In both cases, increasing peptide concentrations led toincreasing cytotoxicity (FIGS. 9B and 9C).

Example 10 Analysis of the Ability of GST-Azu Fusion Peptides to InduceCell Death in Breast Cancer MCF-7 Cells

We have tested the ability of GST-Azu fusion peptides to induce celldeath in breast cancer MCF-7 cells. For measurement of the cytotoxicityof the azurin and plastocyanin synthetic peptides, the3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide) (MTT)(Sigma) assay (Mosmann, J. Immunol. Methods 65:55-63 (1983)). wasconducted. Approximately 2×10⁴ cells per well were seeded into 96-wellculture plates in 100 μl of RPMI 1640 medium. After overnight growth,the supernatant was removed and new media containing azurin orplastocyanin synthetic peptides at various specified concentrations wereadded to the attached cells. After 24 or 48 h treatment, 10 μl of 5mg/ml MTT solution was added to the culture and incubated for 1 h at 37°C. The MTT reaction was terminated by the addition of 40 mM HCl inisopropanol. The MTT formazan formed was measured spectrophotometricallyas described earlier (Mosmann, 1983). Untreated control cells werecompared to treated cells for determining viability and therefore ameasure of cytotoxicity.

There was very little cytotoxicity (FIG. 10) triggered by GST or GST-Azu36-89 fusion protein similar to the control (without protein treatment).However, GST-Azu 36-128 or GST-Azu 88-113, harboring the azurin regioncapable of interfering in ephrinB2/EphB2 binding, showed significantcytotoxicity in a dose dependent manner confirming the role of the Azu88-113 region in triggering MCF-7 cell death.

Example 11 Treatment of Patients Suffering from a Pathological ConditionRelated to Ephrin Signaling

A Phase I/II clinical trial of a cupredoxin compound (Study Drug) isperformed in patients suffering from cancer. Specifically, thecupredoxin compound is Plc 70-84 (SEQ ID NO: 20).

Forty-nine adult patients with histologically verified cancers of thebreast, colon and melanoma who demonstrate clinical and radiographicprogression or recurrence following adequate treatment by currentlyavailable FDA-approved chemotherapeutic drugs and regimen are enrolledin an open-label prospective study administering the Study Drug. To beeligible for enrollment in the study, all patients demonstrateincreasing volume of measurable tumor after completion of approvedcourse of chemotherapy regimens. The evidence of persistent metastaticdeposits and/or continued increase in size or volume must behistologically established. This histological proof can be obtained by afine needle aspiration (FNA) biopsy.

The treatment program is instituted after obtaining informed consentfrom all patients in accordance with the Institutional Review Board ofthe University of Illinois, Chicago and the FDA. The patients will haveno intercurrent illness such as other malignancy, history of previousmalignancy, blood dyscrasias, insulin dependent diabetes or otherserious cardiovascular diseases which might interfere in appropriateevaluation of the effects of the proposed therapy. Baseline blood work(Complete Blood Counts [CBC] and Serum Chemistry) including liverfunction studies (LFT) is performed prior to initiation of therapy. Alleligible patients must not receive any cancer chemotherapy concurrentlyduring the period of the trial.

The study drug(s) is administered by daily intravenous injection of apharmaceutically acceptable preparation of the Study Drug for 12 weeksand the subjects will be observed for any dose limiting toxicity. Therewill be 7 dose levels starting with 10 mg/kg/day and increasing by 5mg/kg/day up to a maximum dose of 50 mg/kg/day. The efficacy of eachdose level will be recorded in 7 patients with advanced measurablecancer (breast, colon, and melanoma).

The response is estimated by measuring the measurable tumor in 2dimensions (a and b). 1) Total disappearance of the target metastatictumors is considered as complete response (CR); 2) A 75% reduction isconsidered excellent, partial response (PR); and 3). A good response(PR) is post treatment reduction in size by 50%. 4) Reduction of 25% insize is considered as stable disease (SD) and 5)<25% is considered as noresponse (NR). Patients demonstrating a progression of disease havetheir treatment discontinued but will be followed for an additional 12weeks.

Total disappearance, and any reduction in size of the target metastatictumors will indicate that the azurin treatment is effective for treatingcancer. Other indications that the Plc 70-84 treatment is effective area decrease rate of in the appearance of new metastatic tumors and adecrease in the angiogenesis associated with tumors.

1. An isolated peptide that is a truncation, wherein said isolatedpeptide is selected from the group consisting of SEQ ID NOS:18, 25, and34 of azurin, can inhibit the growth of mammalian cancer cells in vitro.2. The isolated peptide of claim 1, wherein the azurin is from theorganism Pseudomonas aeruginosa.
 3. A composition, comprising thepeptide of claim 1 in a pharmaceutical composition.
 4. The compositionof claim 3 wherein the azurin is from the organism Pseudomonasaeruginosa.
 5. A kit comprising the composition of claim 3 in a vial.